Breast cancer: insights in disease and influence of drug methotrexate

Methotrexate might be valuable to fight breast cancer.

Abstract

According to the World Health Organization, cancer is one of the leading causes of morbidity and mortality worldwide. The previously estimated 14 million new cases in the year of 2012 are expected to rise, yearly, over the following 2 decades. Among women, breast cancer is the most common one. In 2012, almost 1.7 million people were diagnosed worldwide and half a million died from the disease. Despite having several treatments available, from surgery to chemotherapy, most of these treatments have severe adverse effects. Chemotherapy has a narrow therapeutic window and requires high dosage treatment in patients with advanced-stage cancers and further need innovative treatment strategies. Although methotrexate (MTX) is not a first line drug used against breast cancer, however, it might be valuable to fight the disease. MTX is an effective and cheap drug that might impair malignant growth without irreversible damage to normal tissues. Nevertheless, while MTX does present some disadvantages including poor solubility and low permeability, several strategies are being used to discover and provide novel and effective targeted treatment against breast cancer. In this review, we analyze the chemotherapy of breast cancer and its relationship with drug MTX.

Breast cancer: an overview

According to the World Health Organization (WHO), cancer is one of the major causes of morbidity and mortality worldwide. By 2012 the number of new cases was estimated to be 14 million new cases and 8.2 million deaths1 which is expected to continue rising. In agreement with the previous statement, recent data estimated 18.1 million new cases and 9.6 million deaths occurred in 2018.2 Worldwide, breast cancer, which affects predominantly women, is the most common malignancy and the second leading cause of cancer death mostly in less developed countries.3 Although in rare occasions breast cancer can affect in men or children,3 here we will mainly discuss the breast cancer and its therapeutic strategies in women.

Breast cancer with or without estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER 2) can be divided into hormone-dependent and -independent breast cancer.4 Inside the hormone-dependent breast cancer there is three major subtypes: hormone receptor positive/HER2 negative, HER2 positive and triple-negative (tumor lacks all 3-standard molecular markers.5 Commonly, breast cancer is a hormone-dependent disease and its incidence increase with age. After menopause, the rate of increase incidence is lower (less steep slope) due to the decrease in estrogen concentration (Fig. 1). Throughout the lifetime of a woman, there are three major ages that have the most impact in breast cancer incidence: 1) menarche which is related to a higher risk of breast cancer development; 2) the first full-term pregnancy and the age of menopause. A full-term pregnancy at age of 18 is protecting when compared with women that have never given birth; 3) and late menopause is correlated with higher risk of incidence of breast cancer.6 Overall, the time that a woman is exposed to endogenous reproductive hormones is directly linked with a higher risk of breast cancer development.6

Fig. 1. Breast cancer incidence in women. Average number of new cases per year (bars) and age-specific incidence rates (line) per 100 000 population, females, UK, 2013–2015. Reproduced from ref. 7 with permission from Wiley, copyright 2017.

Additionally, the role of exogenous hormones is also of outer importance since oral contraceptive pills and post-menopausal hormone replacement therapy (HRT) are commonly used. Several epidemiological studies report that the use of estrogen plus progestin contraceptives might increase the risk of breast cancer than estrogen alone.8–11 In 2002, a report by Women's Health Initiative (WHI) trial of estrogen plus progestin, led to a substantial decline of HRT use in post-menopausal women. A 5-year-follow-up study evidences a markedly decreased risk of breast cancer after the discontinuation of combined therapy.12

Besides the exogenous hormones, genetic mutations are also associated with sporadic and hereditary breast cancer. The mutations in the tumour-suppressing genes TP53, BRCA1 and BRCA2 are estimated to account for 30%, 0.7% and 1.3% of cases respectively.13,14 BRCA is a caretaker gene, whose product protein is responsible for the repair of damaged DNA. About 5 to 10% of breast cancers are attributable to either BRCA1 or BRCA2 gene mutation. A single gene mutation is enough for the breast cancer onset.14

Breast cancer treatment

The cancer treatment choice depends on several factors including its stage, tumor location and size, histology and tumor specific properties but also the patient's age (pre- or post-menopause), and pregnancy.15 Taken account this information, it is possible defined a suitable treatment course that are reviewed below.

Surgical and radiation

Surgery remains the core procedure in the treatment of breast cancer and can be a cure when the tumor has not spread to tissues besides the breast and nearby lymph nodes. The patient is submitted to surgery to (1) remove as much neoplastic tissue as possible through mastectomy or breast-conserving surgery (BCS); (2) find out whether cancer cells have spread to the lymph nodes under the arm, by a sentinel lymph node biopsy (SLNB) or an axillary lymph node dissection (ALND);16 (3) restore the breast's shape after tumor removal (aesthetic breast reconstruction);17 or (4) relieve the symptoms of advanced cancer.18 Mastectomy is often chosen by women that might be worried that the less extensive BCS might carrier a higher risk of breast cancer recurrence (Table 1). The information that proves that mastectomy can give a woman any better chance of long-term survival or better outcome from treatment when compared to BCS along with radiation might be scarce.19 However, some studies suggested that the combination of BSC plus radiation could improve the survival, especially the 10-year overall survival.20

Table 1. Mastectomy and BCS features in breast cancer treatment.

	Mastectomy	BCS
Rationale	Safe approach	Aesthetic approach
Procedure	Entire breast removal	Tumor and adjacent breast tissue removal
Early stage cancer	The cure is possible until stage III
Effectiveness	High	High, when combined with radiation
Advanced stage cancer	To delay cancer progression and to provide symptom relief
Effectiveness	Reasonable when combined with radiation and systemic therapy

The removal of adjacent lymph nodes is important for further breast cancer staging and also as part of the therapy protocol (Table 2). Currently, ALND is not an often procedure as it was in the past, yet, in some cases it might be the best way to observe the lymph nodes.21 In cases of advanced breast cancer, a surgery will unlikely cure the tumor that has already spread to other parts of the body. Nevertheless, it could be helpful to slow down the expansion of tumor cells and relieve symptoms. Commonly, the surgery might be used: (1) when the breast tumor is causing an open wound in the breast (or chest); (2) to treat a small number of areas where the tumor has spread (metastases), such as the brain; (3) when the cancer mass is pressing against the spinal cord; (4) to treat a blockage in the liver; (5) or to provide a relief for pain and other symptoms.18

Table 2. Lymph node removal in breast cancer treatment.

Lymph node removal	Sentinel lymph node biopsy (SLNB)	Axillary lymph node dissection (ALND)
Nodes	Few under the arm	Many (usually <20)
Rationale	Where cancer would spread first	Safe approach
Purpose	Cancer staging and therapy

In some women, beside surgery or other treatment, it could be necessary use radiation. The need for radiation depends of many factors as type of surgery the patient had, whether the tumor has spread to lymph nodes or other parts of the body and the patient's age. Tumors with large dimension, or with skin involvement might also require other complementary approaches such as radiation.22 Radiation therapy is a treatment with high-energy rays (such as X-rays) or particles that destroy cancer cells. The two main types of radiation therapy to treat breast cancer are external beam radiation (the body is irradiated by an external machine)23 and internal radiation, also known as brachytherapy (the radioactive source is put inside the body for a short time).24 External beam radiation is the most common type of adjuvant radiation therapy used after surgery. Even so, internal radiation can also be used: (1) in women that underwent BCS, along with external radiation as a way to add an extra boost of radiation to the tumor site; (2) or by itself (instead of whole breast radiation) as a form of accelerated partial breast irradiation (Table 3). The later has the advantage to limit the radiation emitted to the cavity left by the removed tumor, sparing adjacent organs such as the lungs and the heart.25

Table 3. Radiation therapy features in breast cancer treatment. Intracavitary brachytherapy is the most common type of internal radiation therapy currently used. Information compiled from American Cancer Society28.

	External beam radiation	Intracavitary brachytherapy (internal radiation)
Source of radiation	Machine outside the body	Radioactive seeds inside the body
Aimed tissues	Chest wall (after mastectomy), entire breast (after BCS), lymph nodes (if cancer was found), metastasis sites	Breast (where the tumor was removed)
Purpose	Cure delay progression
Requirements	Healed surgery site a , no inflammation, finished chemotherapy	Placement of a device in the space left from BCS
Duration	Few minutes
Schedule	5 days a week (up to 6 weeks)	Twice a day (for 5 days)
Side effects (short-term)	Breast swelling, sunburn symptoms (redness, skin, peeling, darkening), fatigue	Skin redness and bruising (at the treatment site), breast pain, infection, damage to fat tissue
Recommendations	Avoid solar exposition
Complications (long-term)	Breast stiffening and shrinkage, breastfeeding issues, brachial plexopathy, underarm lymph nodes edema, ribs, weakening, angiosarcoma (very rare)	Poor cosmetic results, weakness of the ribs and seroma (fluid collecting in the breast)

^aUnless if it is used intraoperatively after BCS.

Not all women with breast cancer need radiation therapy, but it may be used in several situations: (1) after BCS, to decrease the chance of cancer recurrence in the breast or nearby lymph nodes; (2) after mastectomy, especially if the tumor tissue was larger than 5 cm, or if the tumor was found in the lymph nodes; and (3) if the tumor has spread to other parts of the body such as bones or brain.26,27

Hormone therapy

As previously mentioned, some types of breast cancer are affected by endogenous hormones. Estrogen and progesterone receptor-positive breast cancer cells have membrane proteins that trigger cell growth and division after the attachment of estrogen. Thus, by blocking the estrogen binding to these membrane receptors, it is possible to prevent the tumor development.29 Naturally, hormone therapy is only recommended for women with hormone receptor-positive breast cancers, as it would not help women whose tumors are receptor-negative.29 Usually, is used after surgery as adjuvant therapy to help reduce the risk of relapse. Yet, it can also be started before surgery as a neoadjuvant therapy as well. Similarly, to chemotherapy, hormone therapy is a form of systemic therapy which can be used to treat metastatic breast cancer as well. Since triple negative breast cancers (TNBC) lack hormone receptors, hormone therapy is not effective in the treatment of these types of cancer.30

Several drugs might be used to block estrogen receptors ending endogenous estrogen stimulation for breast cancer cell growth (Table 4). The mainly used estrogen receptor blockers can be subdivided into the following classes: (1) selective estrogen receptor modulators (SERM); (2) and selective estrogen receptor degrader (SERD). These classes of drugs are distinguished based if they correspond to receptor agonists and antagonists, by acting differently across various tissues. Thus, use of these drugs increase the possibility to selectively inhibit or stimulate estrogen-like action in various tissues.31,32

Table 4. Estrogen receptor blockers in breast cancer treatment.

	Anti-estrogenic activity	Estrogenic activity	Indicated to treat	Administration	Bone loss induction
SERM
Tamoxifen	Breast cells	Other tissues (uterus and bones)	All receptor positive breast cancers, including DCIS and metastatic breast cancer; high-risk women a	Oral	No, it may prevent
Toremifene			Metastatic breast cancer	Oral
SERD
Fulvestrant	All tissues	—	Metastatic breast cancer	Buttock injection	Yes

^aWomen with breast cancer family background or BRCA1/2 mutations.

Before menopause, tamoxifen is normally indicated to treat receptor-positive breast cancer, before (neoadjuvant) or after (adjuvant) surgery, and it is usually taken for 5 to 10 years. The advantage of tamoxifen estrogenic activity in other tissues is that it can prevent bone mineral density loss.33 Toremifene is another SERM that acts in a similar way, thus if tamoxifen was previously used and failed or stopped working, toremifene is not likely to work either. The therapy using SERMS might induce side effects that commonly included hot flashes, vaginal dryness or discharge, and mood swings. Fulvestrant is a SERD that acts as an estrogen receptor blocker and degrader. It is used to treat metastatic breast cancer after other hormone drugs (e.g. tamoxifen and aromatase inhibitors) have stopped working. Since fulvestrant block estrogen, its use might cause osteoporosis if taken for a long time.34,35

Following menopause, aromatase inhibitors (AIs) are usually used instead. These drugs block aromatase, an enzyme necessary for the synthesis of estrogen. Letrozole, anastrozole, and exemestane are three AIs that can be taken daily as pills. They can be used in adjuvant therapy after surgery either alone or after using tamoxifen. For most post-menopausal women whose cancers are hormone receptor-positive, AIs are usually recommended at some point of the adjuvant therapy. The standard prescription is to take these drugs for approximately 5 years or to alternate with tamoxifen for a total period of at least 5 years.36

Regard to treatment of breast cancer in pre-menopausal women it is usually recommended to take tamoxifen first, and if the patient goes through menopause during the treatment, they can then take AIs. Together with AI, it can also be prescribed a gonadotropin-releasing hormone analog (GnRH analog) to shut down the ovaries. However, an AI should never be taken alone during breast cancer treatment in pre-menopausal women because it increases gonadotropin levels and, consequently, stimulates follicular growth. AIs can also be used to treat relapsing and metastatic breast cancer, as long as it is hormone positive. The side effects of AIs are usually less severe than tamoxifen, as they do not cause uterine cancers and very rarely cause blood clots. However, muscle pain and joint stiffness, similar to arthritis, may occur in many different joints at the same time. Because AIs drastically lower the estrogen level in post-menopausal women, they can also cause bone thinning, osteoporosis or even fractures. Bone density may have to be periodically check and drugs such as bisphosphonates or denosumab may have to be prescribed in order to strengthen the bones.37,38 In cases of pre-menopausal women with breast cancer, removing or shutting down the ovaries (ovarian suppression) render them post-menopausal which allow use AI hormone therapy (Table 5).39

Table 5. Ovarian suppression in breast cancer treatment.

	Oophorectomy	GnRH analogs	Chemotherapy drugs
Method	Surgical remove	Downregulation of endogenous estrogen production	Ovarian damage
Examples	—	Goserelin and leuprolide	Alkylating agents and platinium salts40
Combinations	—	Alone or together with tamoxifen, Ais or fulvestrant	—
Menopause induction	Permanent	Temporary	Temporary (most of the times)

Targeted therapy

With the advance of biotechnology, the discovery of new vulnerabilities in cancer open avenues to development of novel drugs. Unlike chemotherapy, targeted therapy treatment is specific against cancer cells while chemotherapy may kill normal cells as well. As an example, if a cancer cell overexpresses a growth receptor (GR) and a drug is used to inhibit this GR, normal cells will also be affected by chemotherapy. In contrast, if a cancer cell has a mutated growth receptor that is constitutively active (independent of ligand binding), using a drug that selectively inhibits the mutated protein will not affect normal cells. The only common drawback is that blocking a single pathway in a tumor may be just enough to slow its progression, but it often does not inhibit the cancer enough to eliminate it. Therefore, targeted therapy is normally applied together with traditional chemotherapy.41 About 1 in every 5 women with breast cancer have overexpression of growth-promoting protein HER2/neu. HER2-positive breast cancer cells tend to grow and spread more aggressively than HER2-negative ones.41 Some HER2/neu-targeting drugs have already been developed (Table 6).

Table 6. HER2/neu targeted therapy in breast cancer treatment.

	Type	Early stage cancer	Late stage cancer	Administration
Trastuzumab	Monoclonal antibody	Yes		i.v.
Pertuzumab	Antibody–drug conjugate	Yes
Ado-trastuzumab		—	Yes
Lapatinib	Kinase inhibitor		Yes, usually with trastuzumab and other hormone therapy drugs
Neratinib		Yes, usually after trastuzumab treatment		Oral

The side effects of HER2 targeted therapy are generally mild, but some serious effects may also occur, such as congestive heart failure. The risk of developing heart problems is higher when these drugs are taken together with other chemotherapy drugs that can also cause heart damage, such as DOX and epirubicin. Cardiac function is periodically assessed before, during and after the treatment. Shortness of breath, leg swelling, and severe fatigue are some alarming symptoms of congestive heart failure. Lapatinib and neratinib kinase inhibitors and pertuzumab can cause severe diarrhoea. Moreover, lapatinib can also cause hand-foot syndrome. These drugs may induce birth defects, so they must not be taken when the patient is pregnant, and birth control must be taken during treatment in pre-menopause women.42,43

About two-thirds of all breast cancers express a hormone receptor.44 While treatment with hormone therapy is often helpful, using certain targeted therapy drugs can improve hormone therapy.41 Cyclin-dependent kinase (CDK) inhibitors such as palbociclib, ribociclib, and abemaciclib slow down the cancer progression by blocking cell division, particularly inhibiting CDK4 and CDK6.45 These drugs are approved for women with advanced hormone receptor-positive, HER2-negative breast cancer and are taken once or twice a day as pills. CDK4/6 inhibitors are normally used: (1) together with an AI (or fulvestrant) to post-menopause women; (2) palbociclib or abemaciclib can be given with fulvestrant in pre-menopause women. However, a GnRH analogue to suppress ovaries must also be used; (3) ribociclib can be given with an AI and an ovary suppressor in pre-menopause women.46 CDK inhibitors side effects tend to be mild, being low blood cell counts and fatigue the most common ones. Nausea, vomiting, mouth sores, hair loss, diarrhea, and headaches are less common side effects.47

Olaparib is a poly ADP-ribose polymerase (PARP) inhibitor drug, used against BRCA gene mutation breast cancer. PARP is another protein responsible for the repair of DNA and genomic stability. Because tumor cells with a mutated BRCA gene already struggle in DNA damage repair, blocking PARP proteins often leads to the death of these cells. This drug can be daily taken as a pill to treat metastatic HER2-negative breast cancer in women with a BRCA mutation who have already gotten chemotherapy.48,49 Olaparib can be associated with hormone therapy if the cancer is hormone receptor-positive. The common side effects include nausea, vomiting, diarrhea, fatigue, loss of appetite, taste changes, anaemia, belly pain, muscle, and joint pain. In rare occasions, treatments with a PARP inhibitor have caused the development of a blood cancer, such as myelodysplastic syndrome or acute myeloid leukemia (AML).49

Treatment according to breast cancer stage

The stage of a patient's breast cancer is an important factor in the decision of treatment options. Generally, in cases of extensive spread of breast cancer, it is more likely that its treatment need to be more extensive.5 Other factors that play a role in the treatment decision are the presence of hormone receptors, the presence of large amounts of HER2 protein, the patient's personal preferences, whether the woman has gone through menopause, and how fast the cancer is growing.5 A summarized description of breast cancer treatment strategies is presented in Table 7. Lobular carcinoma in situ (LCIS) used to be categorized as stage 0, however it is no longer considered a pre-malignancy. As this condition increases the risk of developing breast cancer, LCIS should be periodically monitored and the patient should discuss with the physician whether any treatment is needed. In ductal carcinoma in situ (DCIS), mastectomy prevents the contralateral development of breast cancer, and it is recommended for high-risk women (e.g. strong family history and BRCA1/2 mutations). In stage I treatment, radiation can be skipped for some women over 70 years old, as it may not contribute to their longevity (the risk of complications can overweight the treatment benefits). Regard to stage II, the treatment is similar to stage I. The treatment of stage III breast cancer can either start with a neoadjuvant therapy to shrink tumor size or directly to surgery. Inflammatory breast cancer (IBC) that has not spread beyond nearby lymph nodes is also considered to be stage III, but the treatment can be slightly different. IBC is an uncommon type of invasive breast cancer, whose symptoms are caused by the blockage of lymph vessels in the skin. As an IBC has already reached lymph vessels and causes skin changes, it is at least stage III breast cancer. Stage IV (metastatic) breast cancer treatment relies mainly on systemic treatment.50–52

Table 7. Cancer stage and treatment. LCIS – lobular carcinoma in situ; DCIS – ductal carcinoma in situ.

Cancer stages	Features	Treatment	Therapy	Systemic
Stage 0	Limited inside the milk duct, non-invasive
LCIS a	Abnormal cells inside breast lobes, difficult to detect (often during breast biopsy)	Should be discussed with the physician	—	—
DCIS	Abnormal cells inside breast ducts	Lumpectomy, mastectomy, or double mastectomy	May be needed	Hormone therapy
Stage I	Relatively small; only a tiny portion of sentinel lymph node affected (or none)	Mastectomy or BCS; SLNB or ALND	With BCS	If >1 cm
Stage II	Already spread to a few nearby nodes	Similar to stage I	With BCS	Neoadjuvant therapy
Stage III	>5 cm; or invading nearby tissues (breast epidermis or pectoral muscle); or many lymph nodes affected	Mastectomy or BCS; ALND	Needed	Neoadjuvant therapy
Inflammatory (stage III)	Uncommon invasive cancer; abnormal breast skin appearance (thick and pitted like an orange peel); inflammation symptoms b	Mastectomy and ALND after neoadjuvant therapy; surgery is not an option if inflammation is present b or the tumor fails to shrink	If systemic fails to shrink	Neoadjuvant therapy
Stage IV (metastatic)	Spread to other organs (usually bones, liver, and lungs)	Used as local treatments for symptom relief (e.g. open wounds, brain metastases, spinal cord pressure)	Needed (possibly a combination of the three types)	Spread to other organs (usually bones, liver, and lungs)
Stage IV (inflammatory)	Inflammatory; has spread to other organs	Local surgery or radiation for symptom relief	Needed (possibly a combination of the three types)	Inflammatory; has spread to other organs

^aNo longer considered a malignancy.

^bWarmness, redness and swelling.

Sometimes, even years later, it can occur relapse or recurrence of cancers that were previously treated. The recurrence can be local in the breast, or in a distant area as metastases.53 The treatment will depend on which type of recurrence the patient suffers. If a woman has gone through BCS before, a local relapse is usually treated with mastectomy. If the patient was previously subjected to a mastectomy, a local relapse will be, whenever possible, surgically removed and followed by radiation therapy. While regional recurrence may benefit from using systemic drugs as an adjuvant therapy, a distant metastatic recurrence will have to be treated in a similar manner of a stage IV breast cancer.54 In triple-negatives breast cancer, cells grow and spread faster than most other types of breast cancer because they do not need a ligand binding for activation of proliferation signalling pathways. Hormone and HER2 targeted therapies are not helpful for the treatment of triple-negative breast cancer, leaving chemotherapy as the only viable treatment.29 Since there are not many treatments available for this type of breast cancer, the patient should consider taking part in a clinical trial to test an innovative treatment.54

A pregnant patient will have a limited access to treatment options of breast cancer, for the protection of the baby. Treatment choices can be very complex in order to ensure both mother and baby's well-being. For instance, if a woman is diagnosed with breast cancer early in her pregnancy and chemotherapy is straight needed, she may be advised to think about ending the pregnancy, which is emotionally harmful. An early-stage cancer may be surgically treated with either mastectomy or BCS, but the additional radiation therapy needed for BCS is only possible if a woman is already soon to deliver (Table 8). Delaying radiation therapy too long may increase the risk of cancer relapse, thus women in early pregnancy would better choose mastectomy instead of BCS followed by radiation.55

Table 8. Pregnancy and breast cancer treatment.

Trimester	Surgery	Radiation	Chemotherapy	Hormone	Targeted
First	Safe	Harmful	Harmful	Harmful	Harmful
Second			Safe a
Third
After birth	Safe

^aNot recommended after 35 weeks, or within 3 weeks of delivery because it lowers the mother's blood cell counts, increasing the risk of blood loss and infection during birth.

Breastfeeding is generally not recommended in women diagnosed with breast cancer and in need of treatment. For surgery, stopping breastfeeding reduces the blood flow to the breast leads to size reduction making the surgical procedure easier and more effective.56 Furthermore, the risk of infection is lower and avoids having breast milk collected in biopsy or surgery areas. Systemic drug therapies may enter the breast milk and be transferred into the baby during breastfeeding. Thus, if a patient plan breastfeed it is highly recommended to start it after chemotherapy, hormone or targeted therapy have ceased for a while.56

Chimeric antigen receptors (CAR)-T therapy

Treatment using genetically modifying T cells with chimeric antigen receptors (CAR) has been considered to use against breast cancer. The cells are cultivated in the laboratory and then administered to patients by infusion. The new cells have the ability to bind to an antigen presented in the cancer cells and kill them.57 The success of this treatment is dependent on the capacity of infused CAR-T cells proliferate at high levels, thus ensuring that enough cells are available to induce effector function on the encounter with the antigen-expressing tumor.58 This is also one of the major challenges of this treatment. Efforts to improve the number of CAR-T relies on cytokines that are used to culture T cells (reviewed in ref. 57). The function of CAR-T cells may be enhanced not only by addressing stimulatory signals (costimulation, cytokines/cytokine receptor) but also by blocking down regulatory signals.57

Despite the benefits, the strategy might induce potentially severe side-effects including toxicity induced by CAR-T and systemic inflammatory response syndrome (SIRS).59 Nevertheless, there are some strategies including an accurate selection of antigens, infuse CAR-T cells, and activate suicidal genes, as reviewed by Wang and Zhou,57 that could be applied to overcome these drawbacks.

This strategy has been studied either in vitro and in animal models where evaluated several receptors as HER-2, Lewis Y, mesothelin, folate receptor alpha (FR-α), and Muc1.59–66 Nowadays, clinical trials are ongoing using CAR-T cells against breast cancer targeting a cleaved form of MUC1 or targeting HER2-positive.67–69

Chemotherapy and its side effects

Chemotherapy is a type of systemic treatment that uses agents to kill cancer cells or retard their proliferation. Generally, it is administrated orally or intravenously (i.v.) allowing the drugs travel through the bloodstream and reach cancer cells in most parts of the body.70 As mentioned, chemotherapy can be administered as 1) adjuvant, after surgery, in order to prevent relapse of tumor or 2) as neoadjuvant before surgery to shrink tumor until the size gets resectable and can be applied to locally advanced tumors.71 Additionally, neoadjuvant chemotherapy might give valuable information to the physicians regard to chemotherapy administered followed surgery.71 There are many drugs used against breast cancer (Table 9), and in most cases, chemotherapy is most effective when drugs are combined.72 Nowadays, different combined chemotherapy is in clinical practice but thus far, is not clear there is any single combination proven to be the best.

Table 9. Chemotherapy drugs in breast cancer treatment. The drugs presented in this table are the most frequently used drugs in adjuvant and neoadjuvant therapy, as well as in advanced cancer.

Class of drugs	Adjuvant and neoadjuvant therapy	Advanced cancer	Examples
Taxanes	Yes	Yes	Paclitaxel, docetaxel
Anthracyclines	Yes	Yes	Doxorubicin, pegylated liposomal doxorubicin, and epirubicin
Platinum agents	Yes	Yes	Cisplatin and carboplatin
Other agents	—	Yes	Vinorelbine, capecitabine, gemcitabine, ixabepilone, and eribulin
Other agents	Yes	—	5-FU, cyclophosphamide
Employment method	In combination (2 or 3 drugs)	Commonly as a single agent a	—

^aFew exceptions such as paclitaxel plus carboplatin combination to treat advanced breast cancer.

Usually, the administration of chemotherapeutic drugs for breast cancer is intravenously as intramuscular injection, intravenous push technique or intravenous infusion.73 Chemotherapy is given in cycles of 2 or 3 weeks, with each period of treatment followed by a rest period to allow the patient time to recover from the effects of the drugs. Adjuvant and neoadjuvant chemotherapy are often given for a total of 3 to 6 months, depending on the drugs used. The length of the treatment for advanced breast cancer is based on the effect of treatment and its side effects.74 Dose-dense chemotherapy which consist in administration of drugs more often but at same dosage, might lower the chance that the cancer relapse and improve the survival for some women.75 Nevertheless, this therapy present some disadvantages and side effects that depend on dose and length of treatment. Usually, the side effects cease when the treatment is finished.76 Permanent heart damage can be caused in rare occasions by drugs such as doxorubicin (DOX) and epirubicin (EPR). The risk is higher if the drug is used for a long time, in high doses, or combined with HER2 inhibitors (which can also cause heart damage). The cardiac function must be monitored during all stages of chemotherapy, nonetheless, some signs of damage only appear months or years after the treatment stops.77 Nerve damage may be caused by most of the available chemotherapy used to treat breast cancer. Damage to nerves outside the brain and spinal cord can lead to symptoms such as numbness, pain, burning or tingling sensations, sensitivity to cold or heat and weakness. These symptoms appear first in the hands and feet and, in most cases are reversible after chemotherapy.78 Drugs as capecitabine and liposomal DOX can induce the hand-foot syndrome. The symptoms can gradually improve after lowering the drug dose or by the end of treatment.79

Changes in menstrual periods are a common side effect of chemotherapy in younger women and ultimately, premature menopause and infertility may occur permanently.80 These effects are more likely in older women. Usually, is accompanied by increased risk of bone mineral density loss and osteoporosis.81 Nonetheless, there are medicines that can help to prevent bone loss problems.81 Despite chemotherapy might cease menstruation during the course of treatment, it is still possible getting pregnant after that period. It is highly recommended that pregnancy did not occur during treatment since can lead to birth defects and might interfere with treatment.82 In case of diagnose breast cancer during pregnancy it is possible to receive treatment as already described above.

Very rarely, certain drugs can cause bone marrow diseases, such as myelodysplastic syndromes or even acute myeloid leukemia (AML) and usually occurs within a decade after treatment.83 Nonetheless, for most women, the benefits of chemotherapy in helping breast cancer relapse prevention or in extending lifetime are far likely to outweigh the risk of leukemia complication.

As described, chemotherapy has severe adverse effects due to their lack of specificity.84 Cytotoxic effects towards normally proliferating cells and drug resistance acquisition by cancer cells are common reasons for the inefficiency of chemotherapy and ultimately lead to the patient death.85 Since the development of novel drug is very time-consuming and cost-worthy86 it is necessary search for new alternatives. Alternatives might include drug repurposing or drug development based on the conjugation of already existing drugs with other molecules seeking the improvement of bioavailability, specificity, and stability of the active ingredient, which is also known as development of a Drug Delivery System.87 One promising candidate for drug repurpose against breast cancer is methotrexate (MTX) that along with new derivatives could be valuable to combat the disease as presented below.

Methotrexate: an alternative drug for breast cancer?

The drug methotrexate (MTX), formerly known as amethopterin, is an antifolate that acts as an antineoplastic agent and a suppressor of the immune system. Beyond cancer treatment, MTX is used for the treatment of rheumatoid arthritis,88 psoriasis89 and abortion.90 The current clinical applications in cancer treatment include acute lymphoblastic leukemia, medulloblastoma, osteosarcoma, leptomeningeal metastases, gestational trophoblastic tumors, bladder cancer.91,92

Methotrexate is considered as an essential drug in oncology,93 and it also stands in the 20th World Health Organization Model List of Essential Medicines.94 The thought of developing folate analogues for cancer treatment came with observations, such as the administration of folic acid (FA) conjugates to patients with AML resulting in aggravation of the disease.95 Biochemist Yellapragada Subbarao is credited for being the first to synthesize folic acid and to develop its analogues aminopterin and amethopterin (MTX). In 1948, it was reported the use of aminopterin to treat leukemia in children,96 and later MTX for AML treatment.97 MTX eventually replaced aminopterin, for showing a larger therapeutic margin. Curiously, MTX was the first drug to cure a tumor (choriocarcinoma in 1956) in single-agent therapy.98

MTX is not a first line drug to combat breast cancer but there are already several clinical trials underway to improve its effectiveness,99 some involving combinations with other drugs.

In fact, MTX can be used against breast cancer in woman and man, in patients whose cancer has metastasized (spread to other parts of the body) and in women whose cancer was treated with surgery and radiation therapy. For estrogen-negative/androgen-positive breast cancer, MTX is used in combination therapy with cytoxan, or 5-FU.91,92 The drug is used to decrease the chance of invasive breast cancer in patients who have had surgery and radiation therapy for ductal carcinoma in situ.99 Here, we intent understand the dynamics of cancer treatment and whether MTX can be applied with new guidelines against breast cancer.

Pharmacokinetics

MTX is a Biopharmaceutical Classification System class III drug and a weak dicarboxylic acid (pK_a = 4.7–5.5). This drug presents a low permeability (clog P = 0.53) and a poor aqueous solubility (0.01 mg ml^–1).100 Some studies demonstrated that its solubility could be improved by 30-fold through the interaction of MTX with beta-cyclodextrin in the presence of triethanolamine.101 Its absorption mainly occurs in GI tract via proton-dependent active transport which is partially shared with folic acid.100 The dose of MTX administered depends on the of disease for which it is used. For example, the dosage of MTX administered might be either low by oral route for the treatment of rheumatoid arthritis (from 7.5 mg) or very high by i.v. route for the treatment of osteosarcoma (12 g m^–2).102 The plasma levels of drug are higher with i.v. administration, yet, intranasal administration allows higher concentrations in the cerebrospinal fluid (CSF).103 The administration of lower doses leads to a higher bioavailability (42% for dose <40 mg m^–2) while higher dose translate in lower bioavailability (18% for dose >40 mg m^–2).104 The therapeutic and toxic plasma concentration also varies depend on dose of MTX administrated. The higher concentration is register at 24 h follow administration of higher dose.105

Due to erratic gastrointestinal absorption is recommended that MTX might also be administrated using different routes. In the management of rheumatoid arthritis, oral and subcutaneous administration of MTX can be used,88 but intramuscular administration is reported as the most clinically effective with fewer side effects.106 Less invasive ways of administration of drug have a better patient compliance and can be used when the target plasma level of the drug is not very high. On the other hand, most anticancer drugs are administered through the i.v. route.107 MTX is one of the few cancer drugs that needs regular plasma levels monitoring in order to avoid toxicities. Its plasma half-life (t_1/2) ranges from 2 to 10 h. Administration via i.v. provides a better prediction of the MTX plasma levels over time.108

In blood circulation, MTX binds to albumin (K₁ = 820 M^–1) with two binding sites.109 Combination of MTX with other drugs depresses the MTX binding to albumins. In healthy patients, the percentage of MTX bound to proteins (about 50%) is higher than in cancer patients (30%).110 The MTX binding to albumin is dependent on the protein level. Moreover, at high concentrations of MTX, the binding to protein starts being non-linear, increasing disproportionally the plasma levels of free MTX.111 Distribution of MTX to central nervous system (CNS) is a challenge, particularly for the treatment of acute lymphoblastic leukemia, non-Hodgkin lymphomas, and brain tumors.112 MTX is administered by intrathecal route combined with systemic treatment in the prevention of CNS recurrences in acute lymphoblastic leukemia.113 For the treatment of non-Hodgkin lymphomas and brain tumors (medulloblastoma) in children, methotrexate is given by i.v. injection at high doses. The penetration in blood–brain-barrier BBB is weak (1.7–3%) but is clinically enough in childhood medulloblastoma and doesn't seem to be dependent on the drug dosage.114

Metabolism and glutamylation

Folylpolyglutamate synthetase (FPGS) can catalyse the poly-γ-glutamylation of methotrexate (Fig. 2), aminopterin and folate, the naturally occurring molecule.115 The 7-hydroxy metabolites of both MTX and aminopterin are also substrates of FPGS.116 The γ-glutamylation is an important mechanism for the intracellular build-up of folates and improvement of their affinity towards target enzymes, including DHFR inhibition.117 The products of FPGS are not substrates for cellular efflux mechanisms,118 and they allow the establishment of an intracellular pool of MTX that can also be released over time. However, γ-glutamylation is also responsible for hepatotoxicity, due to longer retention times in hepatocytes compared to free MTX (Fig. 2).119

Fig. 2. Proposed methotrexate metabolism pathway. GGH – γ-glutamyl hydrolase; FPGS – folylpolyglutamate synthetase; AO – aldehyde oxidase; CPDG2 – carboxypeptidase G2; GT – glucuronosyl transferase. Reproduced from ref. 120 with permission from ASPET, copyright 2000.

It is not usual to find a glutamylation of MTX longer than triglutamate by FPGS,121 which contrasts with the tetrahydrofolate or folinic acid long-chain polyglutamates, often observed. Consequently, we suggest that early polyglutamates of MTX must bind to FPGS, inhibiting its activity, while only pentaglutamylated tetrahydrofolate is reported to be a good inhibitor.122 The loss of polyglutamates can be performed by a mammalian carboxypeptidase known as γ-glutamyl hydrolase (GGH), yielding back free MTX (Fig. 2).123 Removal of the terminal glutamate in folates is also possible by a class of bacterial carboxypeptidases, which ultimately yields glutamate and 4-amino-4-deoxy-N10-methylpteroic acid (DAMPA) (Fig. 2). It is thought that follow MTX excretion into the gastrointestinal (GI) tract, gut bacteria hydrolyse MTX into DAMPA, which is afterward reabsorbed back into the blood.124 DAMPA cytotoxicity was assessed using Molt-4 human leukemia cell line, and it has shown to be non-cytotoxic and to not affect the MTX toxicity towards these cells.120

Aldehyde oxidase (AO) is the enzyme responsible for the oxidation of MTX into 7-OH-MTX and possibly of DAMPA into OH-DAMPA (Fig. 2).120 The conversion is a process that is independent of CYP450 enzymes. This cytosolic molybdoflavoprotein relies on molybdenum and on flavin adenine dinucleotide (FAD) for the catalysis. It is primarily found in the liver, but it is also present in excretory organs such as lung, the GI tract, and kidney. Along with CYP450 enzymes found in microsomes, AO is responsible for metabolizing drugs and xenobiotic introduced in the body.125 Although 7-OH-MTX and 7-OH-MTX-(Glu)_n metabolites are DHFR inhibitors,126 they also represent a mechanism of toxicity in high dose MTX therapy (HDMTX). The solubility of 7-OH-MTX is about 3–5 fold lower than of MTX at a pH range of 5–7,127 which can lead to crystallization in the nephrons, and consequent delay in renal elimination.95 Similarly to MTX, polyglutamates of 7-OH-MTX maintain a steady-state intracellular pool of this toxic metabolite. Common indications to reduce the risk of nephrotoxicity include alkalinisation and hydration.128

Recombinant carboxypeptidase G2 (CPDG2) from a pseudomonas strain can be administered to patients with HDMTX-induced renal disfunction as a fast rescue agent. Although DAMPA is normally a minor metabolite of MTX, CPDG2 co-administration can yield DAMPA plasma levels close to the amount of infused MTX. CPDG2 can reduce MTX plasma levels by >98% within minutes after the enzyme administration, without any frequent or severe side effects. Furthermore, leucovorin rescue can still be maintained if its dose is increased since CPDG2 can also inactivate leucovorin. OH-DAMPA, OH-DAMPA-glucuronide, and DAMPA-glucuronide metabolites were also identified in plasma and urine when CPDG2 was infused after MTX administration. Enzymatic studies suggest the involvement of AO and CPDG2 in the catalysis (Fig. 2).120

MTX is internalized by cells mainly through reduced folate carrier protein (RFC1) and, to a lesser extent, by α, β and γ folate receptors (Fig. 3).129 The RFC1 plays a key role in the cellular uptake of MTX in excretion organs, and detailed information on membrane efflux transporters are also described in Table 10.

Fig. 3. Overview of MTX pharmacokinetics and pharmacodynamics. Cellular uptake of MTX is mediated by RFC1 and other folate receptors. MTX efflux is mediated by active transporters of the ATP-binding cassette (ABC) family. Inside cells, folylpolyglutamate synthetase (FPGS) catalyzes the glutamylation of MTX, whereas γ-glutamyl hydrolase (GGH) removes glutamate residues from MTX. The intracellular targets of MTX and polyglutamylated MTX (MTX-(Glu)_n) are DHFR, thymidylate synthase (TYMS) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (ATIC). Reproduced from ref. 51 with permission from Multimed Inc., copyright 2019.

Table 10. Main methotrexate membrane transporters. OATP – organic-anion-transporter polypeptide; MRP – multidrug resistance-associated protein; BCRP – breast cancer resistance protein; RFC – reduced folate carrier; OAT – organic anion transporter.

	Transporter	Location	Function
Liver	OATP1B1, OATP1B3	Sinusoidal membrane	Hepatic uptake
	MRP2, BCRP	Apical side of hepatocytes	Biliary clearance
	MRP3, MRP4	Sinusoidal membrane	Passage from hepatocytes back into the blood
	MRP2, BCRP	Apical side of hepatocytes	Biliary clearance
	RFC1	Ubiquitous	Hepatic uptake
Kidney	MRP2, MRP4, BCRP	Luminal side of renal tubular cells	Renal secretion
	OAT1, OAT3	Proximal tubules	Passage from blood to proximal tubules
	RFC1	Ubiquitous	Renal uptake

Excretion of methotrexate is mainly renal, through glomerular filtration and active tubular secretion,130 and about 8.7–26.0% of excretion is biliary.131 After low-dose administration, MTX typically reaches peak plasma concentrations after 1–2 h, and after about 24 h it has been completely eliminated from circulation.132

Pharmacodynamics

MTX interferes with cell division, specifically during the DNA replication (S-stage), when thymidine nucleotide is needed.133 Neoplasia, autoimmune diseases, and pregnancy are the clinical conditions which MTX most interferes, by compromising rapid cell division. In addition to dihydrofolate reductase (DHFR) inhibition, MTX-(Glu)_n also inhibit thymidylate synthase (TYMS) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (ATIC).133

Methotrexate is an antimetabolite type of agent, being a mimetic of the biological occurring molecule FA. Biologically, folate undergoes two steps of reduction to yield tetrahydrofolate (Fig. 4).

Fig. 4. Folate reduction. NADPH cofactor acts as a reducing agent.

MTX competitively inhibits DHFR, an enzyme, which converts dihydrofolate (FH2) into tetrahydrofolate (FH4) (Fig. 5). Tetrahydrofolate is a cofactor involved in the transfer of one-carbon units, when it is in a reduced state: N5,N10-methylenetetrahydrofolate. The primary source of its one-carbon units is from the conversion of serine to glycine (Fig. 6).135 The transfer of one methyl group is needed for the synthesis of thymidine nucleotides, which in turn, is needed for the synthesis of DNA. The inhibition of DHFR by MTX, consequently inhibited DNA replication (during S phase) and compromise cell division.

Fig. 5. Methotrexate mechanism of action. Reproduced from ref. 134 with permission from Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, copyright 2013.

Fig. 6. A. Folate and methotrexate (4-amino-10-methylfolic acid) 2D structures. Folate derivatives are composed of a pterin ring, PABA (para-aminobenzoate) moiety and a glutamate residue. B. Methylation of tetrahydrofolate. PLP coenzyme facilitates the decarboxylation process of serine.

Binding to dihydrofolate reductase (DHFR)

Methotrexate targets DHFR enzyme and binds to what is thought to be an intermediate conformation in the catalysis of folic acid to tetrahydrofolate.136 The active centre of DHFR is rather accessible and allows the complexation of MTX and the NADPH cofactor, which are represented in Fig. 7.

Fig. 7. Human DFHR complexed with MTX and NADPH. MTX is represented as black balls and sticks. NADPH is represented as white balls and sticks. DHFR is represented in grey surface and interacting residues from DHFR are represented in yellow surface (MTX-interacting) and orange surface (NADPH-interacting) (PDB ID: 1DLS).

MTX interacts with DHFR through hydrogen bonds, salt bridges, and hydrophobic interactions. It is thought that the non-hydrophobic interactions play a role in the substrate recognition by the enzyme, and the hydrophobic interactions help the stabilization of the substrate–enzyme complex.137 The majority of the MTX-interacting residues from DHFR are inside its catalytic pocket, being only 3 exposed to the solvent. This contrasts with the NADPH-interacting residues, which most of them are solvent-accessible (Fig. 7 and 8).

Fig. 8. MTX interactions with DHFR residues. Black dashed lines – hydrogen bonds and salt bridges; green dashed lines – π–π interactions; green solid line – hydrophobic interactions (PDB ID: 1DLS).

When bound to DHFR, MTX aromatic rings are accommodated in the inner core of the enzyme and the carboxylic tail is exposed to the solvent, being this a favourable thermodynamic orientation. This binding presented hydrogen bonds (Fig. 9).

Fig. 9. Hydrogen bonds (cyan dashed lines, except Arg-70) and salt bridges (Arg-70) between MTX and protein (PDB ID: 1DLS).

Although the aromatic rings are within π–π stacking distance, the 3D positioning is not either parallel (sandwich and parallel displaced) or perpendicular (T-shaped) (Fig. 10) to be considered a π–π interaction, as it was demonstrated on Fig. 8. Nevertheless, consideration should also be taken to the fact that these figures are only a representation of the atoms mean positions over the time of X-ray crystallography determination, and that the protein may be flexible enough to adopt other conformations in which π–π stacking interactions are present.

Fig. 10. Hydrophobic and π–π interactions between MTX and protein (PDB ID: 1DLS).

When MTX binds to the enzyme, the conformation the pterin part of molecule adopts is in the opposite direction, when compared to FA. This way, the nitrogen that was supposed to go under hydrogenation in MTX doesn't face NADPH (hydrogen donor) (Fig. 11).

Fig. 11. MTX and FA positioning at the DHFR catalytic pocket. Left: MTX (black) and NADPH (white) (PDB ID: 1DLS). Right: FA (black) and NADPH (white) (PDB ID: ; 4M6K).

Mechanism of resistance

During cancer chemotherapy, it is common that cancer cells acquire resistance to the drug, resulting in decreased effectiveness and increased toxicity. Due to the heterogenicity in a cancer cell population, malignancies are highly capable of adapting to the treatment. The most often reported mechanisms of adaptation to MTX are decreased reduced folate carrier (RFC) expression,138 decreased folylpolyglutamate activity,139 increased DFHR expression140 and increased expression of MDR proteins.141

Multidrug resistance (MDR) proteins are a type of drug efflux transporters, belonging to the superfamily of ABC proteins. Drug transporters are mainly expressed in the intestine, liver, kidney, and brain. These proteins play a key role in the absorption, distribution and elimination of xenobiotics.142 Multidrug resistance protein MDR1 (permeability-glycoprotein) and multidrug resistance-associated protein (MRP) transporters are often associated with the development of antineoplastic drug resistance.143 MDR1 is the most widely studied drug efflux transporter and has a broad range of substrates. There are several P-glycoprotein transporter inhibitors in development to combat drug resistance, although none are approved for clinical use, to this date.144 Nevertheless, it is believed that cellular resistance to MTX is independent to the expression of MDR1.145 The MRP family has several members with different drug specificity, tissue distribution and cellular location. While MRP1, 4 and 5 are ubiquitously expressed and overall contribute to MTX resistance,146–148 MRP2 and 3 are only expressed in the liver, kidney and intestine.143 Studies suggest that MRP2 is involved in the biliary excretion of MTX while MRP3 mediates the drug transport from the liver back into circulation, for posterior urinary excretion.149

Novel MTX formulations and their application on breast cancer

MTX presented several limitations as mentioned in previous sections including poor solubility, short half-life in bloodstream, rapid diffusion throughout the body, and the fact that when administered in high doses might lead to drug resistance and toxicity in normal cells. Therefore, it is necessary development novel MTX derivatives or formulations to enhance efficacy and safety of the drug.

The presence of two carboxylic groups in MTX allows the conjugation of other molecules to form prodrugs. Peptides, proteins, and antibodies are frequently employed in bioconjugation chemistry. Additionally, specific spacers between the drug and the supplementary parts have been used as well.150 A study in 1981 with amidation of MTX, claims that α-substituted compounds are significantly less inhibitory than γ-substituted ones. Moreover, it is stated that α-glutamylated MTX retains the same inhibitory effect of free drug.151 However, a study with α and γ-conjugated tetra-branched peptide conjugated with MTX in MCF-7 and MDA-MB-231 cancer cell lines has shown that both regioisomers have the same cytotoxicity. Nonetheless, NT4-α-MTX was slightly more effective in MDA-MB-231 in comparison to MCF-7 (EC₅₀ 5.81 × 10^–7 M and 1.14 × 10^–6 M). Interestingly, in presence of heparin, the cytotoxicity of derivative decreases in both cell lines but did not affect MTX. The peptide used was NT4, a cancer-selective tetra-branched peptide, that binds to lipoprotein receptor-related proteins (LRP) receptors152 and did not present cytotoxic effect when evaluated alone.151 The effect of α and γ-substitution seems to be dependent on the type of molecule conjugated. A recent study developed a novel set of conjugates using pentaglutamylated MTX (MTX-(Glu)₅) that was attached to the N-terminal of cell penetrating peptides (CPP) via peptide bond. Two different CPPs were used, octaarginine and modified penetratin without methionine [penetratin(desMet)] (Fig. 12). Additionally, it was evaluated if the insertion of spacer linker, GFLG, between MTX-(Glu)_n and MTX-penetratin(desMet) maintain or increase the activity of conjugate in two breast cancer cell lines, MCF-7 and MDA-MB-231. The results differ among cancer cell lines. The studies performed in MCF-7 demonstrated that MTX-penetratin(desMet) presented some moderate activity (IC₅₀ 40.2 μM) while MTX is highly active (IC₅₀ 0.8 μM). Also, it was observed that inclusion of spacer increased the activity of conjugated (IC₅₀ 0.3 μM) being more active than drug alone.153

Fig. 12. Novel derivative of MTX including a CPP, PEG and cleavable linker (GFLG) and its effect at 30 μM against MCF-7 cell line. Reproduced from ref. 153 with permission from Elsevier, copyright 2016.

Regard to results obtained for MDA-MB-231, resistant cell line that lacks folate carrier,154 MTX-penetratin(desMet) presented better activity (IC₅₀ 12.9 μM) than those observed in MCF-7 cells. Additionally, in these cancer cell line MTX-Glu₅-penetratin(desMet) was highly cytotoxic (IC₅₀ 0.1 μM). In same fashion, the inclusion of spacer also increases the activity of conjugates.153 This study showed that artificial glutamylation lowers the cytotoxicity of MTX conjugates, thus the selection of CPP and design of conjugate are important.153 This is possible due to the cancelation of positive charges of CPP, necessary for the recognition and internalization. On the other hand, this modification may be beneficial in cells lacking FPGS. Other study using a different CPP, YTA2,155 present the same evidence reported above, which supports the role of CPPs in overcoming the resistance by lack of transporters. The conjugated, MTX-YTA2 at 1 μM was highly cytotoxic against resistant breast cancer cells MDA-MB-231 (EC₅₀ 3.8 μM).155 In a recent study, our research group developed a MTX conjugated with new CPP hexapeptide (Trp–Val–Pro–Thr–Leu–Lys(NH₂)), a short-length polyethylene glycol polymer (PEG) and enzymatically cleavable linker (GFLG), CPGM (Fig. 13).156 The antineoplastic activity of CPGM was lower (IC₅₀ 23 μM) than MTX alone (0.015 μM) against MCF-7 cell line. Apparently, the introduction of CPP, PEG and GFLG could lead to a decrease of the cytotoxicity which is common factor during conjugation chemistry. Thus, the cytotoxicity observed could be attribute to MTX part and other parts only play a role in drug delivery and controlled release of drug from CPGM.156 Nevertheless, the conjugate maximum effect on the cell viability seems to be slightly superior to drug alone which might be indicate that CPGM could be more effective in high dose therapy.156

Fig. 13. Novel derivative of MTX including a CPP, PEG and cleavable linker (GFLG) and its effect at 30 μM against MCF-7 cell line. Reproduced from ref. 156 with permission from Elsevier, copyright 2019.

Using PEG as a drug carrier, Riebeseel et al.,157 developed a set of conjugates of MTX with PEG presenting different molecular weights ranging from 750 to 40 000. In this study it was demonstrated that the length of polymer did not affect the inhibition of the target enzyme, since all PEG–MTX conjugates had similar IC₅₀ values during DHFR assay. Yet, they presented ∼20 to 1000-fold lower antineoplastic activity in comparison to MTX. This fact could be related to the partial derivatization of the α-carbonyl group of MTX that is crucial for the binding to DHFR.157

The inorganic nanoparticles (NPs) have been employed to improve drug delivery, including gold nanoparticles (GNPs). Their physico-chemical properties including unique shape, size and surface, inert nature, high biocompatibility and non-cytotoxicity158,159 render them interesting particles to use in biology and medicine.160,161 Many studies have been performed either using MTX loaded bovine serum albumin (BSA) capped gold nanoparticles,162 multi-wall nanotubes and MTX163 or nanoencapsulation of MTX.164 The conjugated developed by Murawala et al.,162 Au–BSA–MTX demonstrated higher cytotoxicity against MCF 7 cells almost comparable with an equivalent dose of free drug. Using a drug concentration of 1 μg decrease about 20% of cell viability compared to MTX alone. In this study the authors capped the gold nanoparticles with BSA since albumin could be accumulated in solid tumors, thus, it might be suitable to use for drug delivery. Additionally, BSA is useful to improve the stability, toxicity and pharmacokinetic profile of drugs. In fact, the enhanced activity of conjugated is attributed to the preferential uptake of Au–BSA–MTX particles by MCF 7 cells due to the presence of BSA and the targeting ability of MTX to the overexpressed folate receptors in breast cancer cell lines.159 In other study linking MTX with MWNTs using a variety of cleavable linkers demonstrate that cytotoxic efficacy of conjugates is dependent of the type of cleavable linker used. Once again, the coupling occurred via both α and γ carboxylic acids of the MTX Glu moiety.163 The conjugate MWNTs–MTX using a tetrapeptide linker (Gly–Leu–Phe–Gly) induce 90% of MCF 7 cells. The antineoplastic activity observed might be related with the ability of conjugates to enhance cellular uptake of MTX since MWNTs can cross the plasma membrane and release the drug.163 Recently, MTX was incorporated into multiwall lipid-core nanocapsules (MLNC) and the in vitro cellular uptake and antiproliferative activity was evaluated against MCF-7 cells.164 The derivative MTX-Zn–MLNC–MTX at 0.17 mmol L^–1 demonstrated high incorporation efficiency of MTX. Additionally, MLCN formulations containing MTX showed significantly higher antiproliferative activity in comparison to drug alone. Most likely, the results obtained by this derivative are related to their interaction with folate receptors present in MCF-7 cells. It is important to note that MTX-MLCN did not presented cytotoxicity to non-tumoral cells which indicated that they are selective for cell lines with increase expression of folate receptors.164

Yurgel et al.,165 developed a novel MTX diethyl ester [MTX(OEt)₂] and loaded it into lipid core nanocapsules (MTX(OEt)₂-LCN). The apoptotic effect and cell cycle arrest induced by these derivatives were evaluated against MCF-7 and MDA-MB-231 at 20, 10, and 5 μM concentrations. The results were different in two cell lines. For MCF-7 cells both derivatives presented significantly higher apoptotic rates in comparison to untreated cells. Regard to MDA-MB-231, the derivative loaded LNC presented higher antiapoptotic activity in comparison to solution with free drug. The data obtained indicated that LNC formulation might increase antineoplastic effects improving apoptosis induction in resistant cell lines.165 The diethyl ester was also incorporated into MLNC.166 The MLNC derivatives demonstrated to be more active than MTX(OEt)₂ decreasing MCF-7 cell viability, increase uptake and antiproliferative activity and do not present cytotoxicity against non-tumoral cells.166 Taken into consideration the studies described above, it seems that incorporation of drugs or diethyl ester derivative into nanocapsules is an interesting and promising strategy to improve drug delivery and antineoplastic activity, even against resistant cell lines.

Other strategy that have been study was the incorporation of MTX with chitosan.167 Chitosan is obtained by deacetylation of chitin, a widespread natural polysaccharide found in exoskeleton of crustaceans.168 Their reactive amine ad hydroxyl groups makes this polysaccharide suitable for modifications.165 In study performed by Yu and colleagues,167 developed novel amphiphilic derivatives of MTX–chitosan oligosaccharide (MTX–CHO) with different feeding ratios of drug for sustained release. The release of drug depends on drug loading amount, i.e. the greater of the amount of drug in micelle slow down its release. Both loaded and unloaded micelles demonstrate antineoplastic activity against MDA-MB-231 cell line. For examples, at lower concentration of 1 μg ml^–1, MTX–CHO derivatives significantly decrease the cell viability (45.17 ± 9%) in comparison to viability induced by unloaded micelles (91.86 ± 9.88%). Despite the MTX–CHO derivatives demonstrated cytotoxicity, it was lower than free drug. Nevertheless, because of their properties as colloidal stability, good loading efficiency and sustained release of drug, MTX–CHO micelles might be a promising candidate for drug deliver to cancerous cells.167

As mentioned on previous sections, combine therapy is a common practice in the treatment of cancer and can reduce adverse effect and drug resistance as well increase the efficacy of treatment. Thus, this a strategy that should be pursued and several studies have been performed to find novel chemotherapy combination. MTX and AICA riboside (5-aminoimidazole-4-carboxamide riboside), a cell permeable nucleoside and AMP-activated protein kinase (AMPK) activator,169 was combined and evaluated in MCF-7 cell line and nude mice bearing MCF-7 cell xenografts.170 Combine MTX (0.1, 0.5 and 1 μmol L^–1) with AICA riboside (0.25–1 mmol L^–1) achieve synergistic cytotoxicity in vitro. Similar results were observed in vivo studies were co-treatment with two compounds (MTX 50 mg kg^–1, iv; AICA 200 mg kg^–1, iv) exert significantly higher inhibition on the tumor growth (79.92 ± 34.61%) in comparison to either drug alone (55.00 ± 0.79% MTX and 42.16 ± 12.15% AICA). During in vivo studies it was demonstrated that MTX influence the pharmacokinetics of AICA riboside leading to a higher concentration of AICA riboside and its active metabolite in tumors.170 The metabolic consequences of combine regimen of MTX and AICAR was also assessed in vitro in MCF-7 cell line.171 Despite MCF-7 cells resistance to individual treatment of AICAR and MTX, when drugs were combined lead to a decrease of cell proliferation translated in a reduction in cell numbers approximately ∼50%. Additionally, the combination of two drugs might enhance mitochondrial oxidation and modulate an independent metabolic pathway which could induce synthetic lethality.171 Consequently, the combined regiment might exert a potential cytostatic regime reverting Warburg metabolism which is important since breast cancer cells are known to depend on Warburg-type rearrangement of metabolism.172 Recently, the combination of MTX and vitamin C have been evaluated against triple-negative breast cancer cells (TNBC).173,174 The combination of vitamin C (30 μM to 4 mM) with MTX demonstrated to inhibit the growth of MCF-7 and MDA-MB-231 cell through G2/M elongation and PI3K activation.173 In other study using the combination but with lower dose of vitamin C (5 μM) and MTX achieved synergistic anti-proliferative/cytotoxic effects on TNBC through increasing H₂O₂ levels and activation of caspase-3 and p38 cell death pathway.174 Recently, the combination of MTX with vitamin E variants (α-tocopherol) and derivatives (α-tocopherol succinate) were assessed on TNBC.175 The study demonstrated that combination with MTX and vitamin E variant suppress TNBC proliferation. The cell survival rate of MDA-MB-231 cells was about 35% when treated with MTX (10 μM) and α-tocopherol succinate (40 μM). However, decreasing the concentration of MTX to 0.1 μM, the cell survive rate was above 60% indicating that high dose MTX enhance anticancer activity. The combination of MTX/α-tocopherol succinate is more suitable than MTX/α-tocopherol.175

In order to increase selectivity for cancer cells, Kmiecik et al.,176 developed novel MTX and epirubicin (EPR) conjugates. EPR is a semi-synthetic anthracycline antibiotic derivative of doxorubicin. The drugs were linked through amide bond between MTX carboxylic group and the amine group EPR resulting in the formation of monosubstituted and disubstituted MTX derivatives. Either derivatives were less cytotoxic against cancer cell lines (IC₅₀ 20–35 μM) than free drugs (IC₅₀ > 22 μM for MTX and 0.550 μM for EPR). The lower cytotoxicity of derivatives might be related to the fact that conjugation through γ-carboxylic group of MTX can prevent polyglutamylation and drug uptake177 while modification via α-carboxylic group can interfere with interaction between drug and DHFR.178 Additionally, the antineoplastic activity of EPR might be inhibited follow conjugation with MTX.176 Despite these results authors considered that synthesis of novel MTX–EPR derivatives containing a cleavable linker between the drugs might be valuable to increase cytotoxicity and drug release.

One of the promising strategies to improve efficacy and provide a novel therapeutic solution for breast cancer consists of the use of an antibody–drug conjugate (ADC). ADCs an emerging novel classes of anticancer treatment and uses and antibody-mediated delivery of cytotoxic drugs to the tumors. Thus, this treatment targets the cancer cells and consequently spares normal cells, which might improve the specificity and selectivity.179,180 Thus far, there is one ADC, ado-trastuzumab emtansine (T-DM1) approved by FDA for HER2-positive breast cancer.181 ADCs as trastuzumab-emtansine, and glembatumumab vedotin are in phase III and II trials, respectively, conducted in patients with HER2 positive metastatic breast cancer.182 Since the 1980's antibodies conjugated-MTX have been synthesized and evaluated against different cancers like osteosarcoma, melanoma or non-small cell lung carcinoma, among others.183–188 However, these agents did not achieve clinical benefit due to their moderate cytotoxic potential, lack of selectivity, and low intracellular drug concentration.189,190 Probably, this explained why no antibody–MTX conjugate was evaluated against breast cancer, at least to our knowledge. Nevertheless, this strategy should be pursued, perhaps using a modified and less cytotoxic MTX derivative, since the possibility of a selective and target treatment with the minor adverse effect would be beneficial for patients.

Conclusions

Breast cancer is the common malignancy affecting millions of women worldwide. Several treatments are available according to cancer stage, patient age, type of cancer among other factors. Nevertheless, treatments are most of the times invasive and presented adverse and threatful side effects. More importantly, cancer cells may become resistant to therapy. Therefore, it is necessary pursue the search and development of novel therapy strategies. Despite MTX is not considered a first-line drug for breast cancer, it has clinical importance. Although the drug presented some disadvantages, here we elucidate several strategies including conjugation of MTX with CPPs, nanoparticles or novel derivatives might be useful to improve pharmacological properties of MTX. Additionally, novel MTX derivatives increase its antineoplastic activity and improve its delivery render breast cancer treatment more selectivity reducing cytotoxic effect against normal cells. Thus, it is reasonable hypothesized that using these derivatives might decrease the adverse effects related to treatment and therefore might improve patient's life quality. Importantly, the conjugation of MTX with different molecules had demonstrated that this strategy could be suitable and effective against resistant breast cancer cells. We do believe that the use of MTX and development of novel derivatives using different strategies should continue to be pursue for an innovative and improved breast cancer treatment.

Conflicts of interest

There are no conflicts to declare.