Synergistic anticancer effects of doxorubicin and metformin combination therapy: A systematic review

HIghlights

• •Combined Doxorubicin and metformin regimens exert anticancer effects on various cancers in vitro and in vivo.
• •Combining metformin with Doxorubicin inhibited cell viability and decreased colony formation by inducing apoptosis or sensitization of cancer cells to the Doxorubicin.
• •The synergistic effects of metformin with Doxorubicin could reduce Doxorubicin drug resistance by various mechanisms.

Abstract

Introduction

Doxorubicin (DOX) a chemotherapy drug often leads to the development of resistance, in cancer cells after prolonged treatment. Recent studies have suggested that using metformin plus doxorubicin could result in synergic effects. This study focuses on exploring the co-treat treatment of doxorubicin and metformin for various cancers.

Method

Following the PRISMA guidelines we conducted a literature search using different databases such as Embase, Scopus, Web of Sciences, PubMed, Science Direct and Google Scholar until July 2023. We selected search terms based on the objectives of this study. After screening a total of 30 articles were included.

Results

The combination of doxorubicin and metformin demonstrated robust anticancer effects, surpassing the outcomes of monotherapy drug treatment. In vitro experiments consistently demonstrated inhibition of cancer cell growth and increased rates of cell death. Animal studies confirmed substantial reductions in tumor growth and improved survival rates, emphasizing the synergistic impact of the combined therapy. The research' discoveries collectively emphasize the capability of the co-treat doxorubicin-metformin as a compelling approach in cancer treatment, highlighting its potential to address medicate resistance and upgrade generally helpful results.

Conclusion

The findings of this study show that the combined treatment regimen including doxorubicin and metformin has significant promise in fighting cancer. The observed synergistic effects suggest that this combination therapy could be valuable, in a setting. This study highlights the need for clinical research to validate and enhance the application of the doxorubicin metformin regimen.

Graphical abstract

Introduction

Cancer therapy faces significant hurdles that hinder its effectiveness and success. One prominent challenge is the cancer cells' ability to regulate gene expression and exploit genes from various pathways to sustain their survival and growth [1,2]. This capability stems from genetic mutations, tumor heterogeneity, epigenetic changes, and the presence of cancer stem cells [3]. Moreover, the phenotypic diversity within tumors, resulting from genetic and epigenetic variations, allows subpopulations of cancer cells to adapt to different microenvironments, resist treatment, or facilitate metastasis [4]. Given the diverse strategies employed by cancer cells to ensure their survival and the significant adverse effects associated with cancer chemotherapy, it is imperative to develop comprehensive treatment plans that incorporate multiple approaches to effectively counteract these challenges [5,6]. Combination therapy, which entails the use of multiple treatment strategies, has emerged as a practical approach employed by researchers to overcome these challenges.

Doxorubicin (DOX) is a potent and versatile anticancer agent used to treat a wide range of cancers, including bladder, breast, stomach, lung, ovarian, thyroid, soft tissue sarcoma, multiple myeloma, and Hodgkin's lymphoma [7]. Its mechanisms of action involve DNA intercalation, disruption of mitochondrial function, inhibition of topoisomerase II, and the production of free radicals, resulting in DNA and cell membrane damage [8,9].The pleiotropic nature of doxorubicin enables it to elicit various cellular responses, encompassing cell-cycle arrest, growth arrest, the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), caspase-3 activation, the induction of both extrinsic and intrinsic apoptotic pathways, the activation of pyroptosis, the initiation of senescence, the induction of ferroptosis, and the activation of the AMP-activated protein kinase (AMPK) and C-Jun N-terminal kinase (JNK) signaling pathways [10], [11], [12]. Despite its efficacy, the development of doxorubicin resistance in tumor cells is a significant challenge [13]. Prolonged use of doxorubicin can prompt cancer cells to switch to alternative molecular pathways, contributing to drug resistance. Increasing the doxorubicin dosage to overcome resistance is not advisable due to the associated adverse effects, such as cardiotoxicity and organ damage in the brain, liver, kidneys, and bone marrow [11]. Combining doxorubicin with other cytotoxic agents has become a prevalent strategy to overcome resistance. This approach allows for the administration of lower doxorubicin doses, mitigating adverse effects while simultaneously maximizing therapeutic efficacy and minimizing undesirable effects [14].

Metformin, besides its efficacy in managing type 2 diabetes mellitus, has exhibited strong potential as a complementary treatment option for a range of other conditions, including polycystic ovary syndrome (PCOS), non-alcoholic fatty liver disease (NAFLD), gestational diabetes, and cardiovascular disease. Additionally, recent research has explored its antiviral and anticancer properties [15]. The primary mechanism of action of metformin involves the activation of AMP-activated protein kinase (AMPK), a regulatory protein crucial for cellular metabolism. The current academic consensus on the pharmacological influence of metformin on cellular physiology primarily stems from its AMPK-dependent effects. However, it's important to note that metformin also has independent effects on other cellular pathways [16]. Activated AMPK phosphorylates downstream proteins involved in catabolic processes such as fatty acid β-oxidation, glycolysis, cholesterol biosynthesis, protein synthesis, and fatty acid synthesis [17]. Likewise, metformin inhibits hepatic gluconeogenesis and enhances glucose uptake in muscles, thereby improving insulin sensitivity and reducing fasting blood glucose levels in individuals with diabetes [18].

The AMPK signaling pathway plays a central regulatory role within the cell, exerting significant influences on various cellular pathways, including the Ras/Raf/MEK/ERK pathway, PI3K/AKT pathway, mTOR pathway, Wnt/β-catenin pathway, Notch pathway, Hedgehog (Hh) pathway, transforming growth factor-β (TGF-β) pathway, JNK signaling pathway, PI3K/PDK1/AKT2 pathway, p53-mediated apoptosis pathway, ErbB family pathway, GSK3 signaling pathway, insulin-like growth factor (IGF) signaling pathway, inflammatory signaling pathways (e.g., NF-κB pathway, JAK-STAT pathway, MAPK pathway, TLR pathway), and inflammasome pathway [15,19,20]. These intricate interactions enable AMPK to regulate a wide range of cellular processes related to growth, proliferation, survival, metabolism, inflammatory response, DNA repair, autophagy, and cell cycle progression [21]. Metformin, due to its intertwined mechanism of action with the AMPK pathway, can influence all the cellular pathways that have crosstalk with AMPK [22].

Recent research has demonstrated that standard doses of metformin (1500 to 2250 mg/day in adults) reduce the incidence of cancer and cancer-related mortality in diabetic patients. Notably, metformin's ability to lower blood insulin levels may play a key role in its efficacy against hyperinsulinemia-associated cancers, such as breast and colon cancer [23]. Furthermore, metformin may directly inhibit cancer cells by interfering with the mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis.Numerous epidemiological, preclinical, and clinical studies have provided compelling evidence supporting the successful combination of metformin and doxorubicin in treating various cancers. This systematic review aims to compile and summarize the available studies on the benefits and drawbacks of combination therapy involving doxorubicin and metformin in cancer treatment.

Methods

We conducted a comprehensive and systematic search according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [24]. To ensure a well-organized review process, we employed the PICO framework, which comprises the following elements:

Participants (P): In vivo or in vitro studies involving various types of cancer

Intervention (I): Participants in vivo or in vitro receiving a combination of doxorubicin and metformin

Comparator (C): A group receiving only doxorubicin compared to the group receiving the combination treatment

Outcomes (O): Two key outcomes were assessed:

(1) Cancer cell toxicity and death, including tumor growth, apoptosis, and cytotoxicity

(2) Inhibition of mechanistic pathways, such as colony formation and autophagy

The combined treatment of doxorubicin and metformin resulted in changes in cancer cells and tissue compared to doxorubicin treatment alone.

Search strategy

We conducted a comprehensive and methodical review of relevant published literature using various online databases, including Embase, Scopus, Web of Science, PubMed, ScienceDirect, and Google Scholar, up to January 2023. Table 1 presents the search terms chosen from the Scopus database; the search terms employed in the remaining databases can be found in the supplementary file.

Table 1.

Search terms for the current research based on Scopus.

doxorubicin keywords	AND	Metformin keywords	AND	Cancer keywords
TITLE-ABS(Farmiblastina) OR TITLE-ABS(Ribodoxo) OR TITLE-ABS(Rubex) OR TITLE-ABS (Adriamycin) OR TITLE-ABS(Adriblastin*) OR TITLE-ABS(Adrimedac) OR TITLE-ABS("DOXO cell") OR TITLE-ABS(Doxolem) OR TITLE-ABS(Doxorubicin*) OR TITLE-ABS(Hydrochloride) AND TITLE-ABS(Doxorubicin) OR TITLE-ABS(Myocet) OR TITLE-ABS(Onkodox)	AND	TITLE-ABS(Metformin) OR TITLE-ABS(Dimethylbiguanidine) OR TITLE-ABS(Dimethylguanylguanidine) OR TITLE-ABS(Glucophage) OR TITLE-ABS ("Metformin Hydrochloride") OR TITLE-ABS(Hydrochloride) AND TITLE-ABS(Metformin) OR TITLE-ABS ("Metformin HCl") OR TITLE-ABS(HCl) AND TITLE-ABS(Metformin)	AND	(Tumor [Title/Abstract] OR Neoplasm [Title/Abstract] OR Cancer [Title/Abstract] OR Malignancy* [Title/Abstract] OR "Malignant Neoplasm*"[Title/Abstract] OR (Neoplasm* AND Malignant) [Title/Abstract] OR "Benign Neoplasm*"[Title/Abstract] OR (Neoplasm* AND Benign) [Title/Abstract])

Inclusion and exclusion criteria

All included studies, whether in vivo or in vitro, were relevant to our objectives. To ensure research quality, the search was initially restricted to original articles written in English, excluding letters, review articles, commentaries, and conference papers. The articles meeting this criterion were then carefully reviewed, and relevant studies were identified for data extraction. To ensure accuracy and consistency, two authors independently conducted the literature searches, selected articles, and synthesized data. In case of any discrepancies, the authors collaborated to resolve them before proceeding further.

The study collected and recorded essential data for reporting its results, including information on the article and the type of disease. Additionally, information on the type of animals or cells, the number and mean age of patients (in human studies), dose and duration of therapy, and treatment efficacy were extracted and described. Notably, the study considered animal/cell survival as the most crucial outcome, followed by tumor size and tumor growth inhibition rate. To describe treatment efficacy, the study employed fold change or percentage efficiency compared to the cisplatin-alone group, which were extracted and verified by two independent authors.

Results

Following the application of predetermined inclusion criteria, a comprehensive literature search identified a total of 651 articles. These were distributed across various databases: PubMed (125), Scopus (126), Web of Science (WOS) (176), and others (Embase: 192, Google Scholar: 29). An additional three articles were identified through reference list screening of included articles. After excluding irrelevant documents and review articles, 30 articles were selected for data extraction. The article selection process is illustrated in Fig. 1. For a comprehensive overview of the included studies and the extracted data, please refer to Table 2.

Fig. 1.

Flow diagram of the study selection process.

Table 2.

Key Features of studies included in the systematic review.

NO	Reference	Disease/study type	Patients/country	outcomes	Doses/treatment period	Age	Mechanistic pathways
1	( Barredo JC et al.2016) [37].	Relapsed refractory acute lymphoblastic leukemia/A phase I window, dose escalating and safety trial	A total of 14 patients (10 male and 4 female) were recruited within the period spanning from July 2012 to April 2016. /USA	complete remissions, treatment tolerability, partial response, compliance to the experimental treatment, stable disease, progressive disease and death	It was administered at a dose of 1000 mg/m2 per day, along with vincristine, dexamethasone, PEG-asparaginase and DOX.	1–30 years	AMPK has an impact on the unfolded protein response (UPR), leading to heightened susceptibility to endoplasmic reticulum (ER) stress within cells. Evaluation of pharmacodynamics has revealed the induction of ER stress, the activation of AMPK, and the suppression of the UPR.
2	(Nanni O et al.2018) [38].	HER2-negative metastatic breast cancer (MBC)/ a phase II, open-label, multicenter, randomized clinical trial	126 Non-diabetic women with HER2-negative MBC/ Italy	Overall survival (OS), progression-free survival (PFS),Response and progression,	In arm A, the regimen consisted of non-pegylated liposomal doxorubicin (NPLD) at 60 mg/m2 administered intravenously (i.v.), along with cyclophosphamide (C) at 600 mg/m2 i.v., and Met-at a dose of 1000 mg administered twice daily. In arm B, the treatment included NPLD at 60 mg/m2 i.v. combined with C at 600 mg/m2 i.v. Both arms underwent chemotherapy cycles every 21 days for a total of 8 cycles.	Arm A 57 (50–68) Arm B 61(54–66)	The anticancer effects of Met-are thought to be mediated by the activation of the AMPK pathway or through an indirect influence possibly linked to the modulation of host metabolism, particularly in individuals with insulin resistance.
NO	Reference	Disease	Animal/cell line	Outcomes	Doses/treatment period	Mechanistic pathways
3	(Iliopoulos D et al.2011) [35].	Prostate cancer	female nu/nu mice injected with PC3	tumor growth, prolong relapse	1 mg/kg DOX, Met (200 mg/mL), 55 days	inhibit highly tumorigenic (CSC-like) cells
4	(Iliopoulos D et al.2011) [35].	Lung cancer	female nu/nu mice injected with A549	tumor growth, prolong relapse	1 mg/kg DOX, Met (200 mg/mL), 55 days	inhibit highly tumorigenic (CSC-like) cells5
5	(Kabel AM et al.2016) [36].	Breast cancer	BALB/C mice injected with Ehrlich carcinoma cells (ECC)	Survival rate, tumor volume, Apoptosis	4 mg/kg DOX, Met (200 ug/mL),42 days	Met-was found to reduce the activity of the SphK1 enzyme, thereby enhancing its effect and reducing the resistance of cancer cells to DOX.
6	(El-Ashmawy NE et al.2017) [40].	Mouse breast adenocarcinoma	Mice injected with Ehrlich ascites carcinoma (EAC) cells	tumor volume, survival rate, apoptosis	Met(15 mg/kg) and DOX (4 mg/kg),27 days	Activation of AMPK
7	(Salim E et al.2021) [41].	Breast cancer	mammary carcinogenesis induced female Sprague -Dawley (SD) rats	Mammary tumor latency, Mammary tumor incidence and multiplicities	Met (2 mg/ml) and DOX (4 mg/kg),26 weeks	Inhibition of cellular growth and suppression of mRNA expression related to EGF and ERα.
8	(Li Y et al.2018) [30].	Breast cancer	MCF7/ADR xenografts bearing female nude mice	Tumor volume, tumor weight	DOX (0.5 mg/kg) and Met (100 mg/kg),15 days	Inducing the elimination of cancer cells positive for Ki67 and inhibited the expression of Pgp by Met
9	(Hirsch HA et al.2013) [31].	Breast cancer	female nu/nu mice injected with MCF10A-ER-Src cells	Tumor volume	1 mg/kg or 4 mg/kg DOX, 200 μg/mL Met,20 days	Inhibition of the Inflammatory Feedback Loop.
10	(Hirsch HA.2009) [26].	Prostate cancer	female nu/nu mice injected with LNCaP and DU145 cells	Tumor volume	4 mg/kg DOX, 200 μg/mL Met,20 days	Inhibition of the Inflammatory Feedback Loop.
11	(Hirsch HA.2009) [26].	Breast cancer	female nu/nu mice injected with MCF-10A ER-Src cells	Tumor volume Prolong remission	Intraperitoneal injections every 5 days (3 cycles (25 days)) with 4 mg/kg DOX, ), 100 μg/ml Met, or both.	Met-demonstrates targeted efficacy against cancer stem cells with a distinctive CD44high/CD24low phenotype. When employed alongside DOX, it exhibits the capacity to decrease both nCSCs and CSCs within a heterogeneous transformed population. Moreover, its ability to impede the transformation of MCF10A-ER-Src cells suggests its potential in the prevention of cancer progression.
12	(Shi WY et al.2022) [32].	Lymphoma	nude mice injected with B- and T-lymphoma cell lines	Tumor volume, autophagy	For ip use, (2 mg/kg/day) Met-for 21 days. For p.o use, oral Met (3 mg/kg/day), ip DOX (3 mg/kg/day, twice weekly)	Activation of AMPK, leading to the targeting of the mTOR pathway, induces autophagy in lymphoma cells.
13	(Zhang H et al.2022) [25].	Breast cancer	female nu/nu mice injected with transformed MCF10A-ER-Src cells	Tumor volume	4 mg/kg DOX (dox), 100 Ag/mL Met,15 days	The capacity of Met-to selectively eliminate cancer stem cells is enhanced when used in conjunction with DOX, effectively obstructing both cancer stem cells and transformed nCSCs in a synergistic manner.
14	(Zhang H et al.2022) [25].	Breast cancer	MCF-7/DOX tumor-bearing mice.	Tumor volume	(1 mg/kg) of free DOX., lipid/PolyMet-(PGA-DOX) nanoparticles,15 days	PolyMet has the capability to effectively impact the expulsion activities and inhibit the expression of ABC transporters (ABCB1 and ABCC1), thereby overcoming DOX-related multidrug resistance.
15	(Banerjee I et al.2019) [34].	Breast cancer	MDA-MB-231 xenograft model	Tumor volume	Met-was administered at a dosage of 150 mg/kg body weight daily, while PLD (pegylated liposomal DOX) was administered with metronomic dosing at 1 mg/kg body weight in relation to DOX, every other day for a duration of 3 weeks.	The significant anticancer effects observed in vitro and in vivo upon the combination of Met-and PLD can be attributed to the down-regulation of β-catenin.
16	(Zheng A et al.2007) [29].	Breast cencer	Tamoxifen induced MCF-10A Er-Scr cells and MCF-7 cells	Cell viability	DOX and 0.1 mM Met, 48 h	_ Met-intervenes in the insulin/IGF signaling pathway and the AMPK pathway, thereby stimulating the AMPK/mTOR pathway.
17	(Chen G et al.2012) [33].	Thyroid cancer	HTh74 and HTh74Rdox cells	Cell viability, Cell cycle arrest, apoptosis, Inhibition of colony and sphere formation	DOX [50 ng/ml, Met [5 mM,48h
18	(Qu C et al.2014) [42].	Breast cancer	MCF7, MCF7/5-FU, and MDA-MB-231	Cell invasion, cytotoxicity, apoptosis, growth rate	Met 0.75 mg/L, 10 uM Met 72h	Through the activation of AMPK, it is capable of reversing both MDR and EMT.
19	(Cooper AC et al.2015) [43].	Breast cancer	BT474, MDA-MB-453, SKBr3, MDA-MB-468	Cytotoxicity	DOX 200 nM (for BT474 cells 500 µM) and Met (10 mM) for 72 h	The activation of AMPK was observed in breast cancer cell lines that exhibited overexpression of HER1 and HER2.
20	(Sliwinska A et al.2015) [44].	Hepatocarcinoma	HepG2 cells	Cell viability apoptosis	DOX 50Nm Met 5Mm 72 h for mtt 48 h for apoptosis	Met-was found to enhance the toxicity of WP 631 in HepG2 cells through the upregulation of NF-jB and the downregulation of p53.
21	(Wu X et al.2017) [27].	gastric cancer	AGS Cell line	cell proliferation, migration, apoptosis	Met 10 mM, 0.02 mg of Adriamycin (DOX) 48h	Activation of AMPK
22	(Candido S et al.2018) [45].	Pancreatic ductal carcinoma	MIA-PaCa-2 cells / ASPC-1 cells	Cell viability	Met 250 nM and Met 90 nM, 48h	Activation of AMPK
23	(Shafiei Irannejad et al. 0.2018) [46].	Breast cancer	MCF-7/DOX	Apoptosis cytotoxicity	Met 10 mM, Met 3.23 uM, 48 h	Reducing P-gp activity
24	(Gatti Metal 0.2018) [47].	Osteosarcoma	CSC osteosarcoma 2(OSA2)	Cell viability Cell invasion	0.2 uM DOX and1.8 mM Met, 48 h	Suppression of ABCB1 activity, cell cycle arrest at the G1/S transition, and initiation of apoptosis.
25	(Shafa MH et al.2018) [28].	Prostate cancer	DU145 cancer cells	Cytotoxicity, cell cycle arrest, apoptosis	DOX 0.0005 (μM), Metf 10 (mM),48h	Blocking the function of ABCB1, increased expression of p21, leading to cell cycle arrest at the G1/S transition, and initiation of apoptosis.
26	(Paiva-Oliveira DI et al.2018) [48].	Osteosarcoma	Spheres of MNNG/HOS and MG-63 osteosarcoma cell lines	Cytotoxicity, cell viability	DOX (0.25 Um), Met (5 mM), 48 h	Activation of AMPKα signaling pathway
27	(Mohammd AW et al.2019) [49].	Breast cancer	MCF-7 cell lines	Cytotoxicity, proliferation arrest, apoptosis	DOX5 uM and Met 10 mM 72 h	Met-exhibits a cytoprotective effect, reducing both basal and postprandial glucose levels through the attenuation of ROS production. This effect is achieved by the maintenance of energy homeostasis and regulation of apoptosis through its activation of AMPK.
28	(Lee ACK et al.2021) [50].	Hepatocelular carcinoma (HCC)	cancer stem cell (CSC) R-HepG2	Cytotoxicity	2 mM Met, 20 uM DOX 24 h	Met-obstructs the activity of mitochondrial ETC Complex I, leading to a decrease in NADH oxidation.
29	(Agnieszka Mlicka et al.2023) [51].	Bladder cancer	T24 cell line	Cell viability,colony formation and migration	DOX and Met-in the 500:1 ratio (500 μM MET / 1 μM DOX) for 24 h	Activation of AMPK
30	(Meyer FB et al.2021) [39].	Breast cancer	BALB/c fibroblasts (BALB/c-3 T3-A31–1–1 cells)	Cell viability	Treatment with 1 and 10 nM DOX individually did not yield any significant impact on type III foci. However, when combined with 1 mM Met-in a concurrent treatment, there was an observed increase in the number of type III foci by 14 % and 18 % at 72 h.	_

The analysis included a majority of in vitro studies (14 articles) investigating the combination of doxorubicin and metformin for cancer therapy. Fourteen additional studies employed animal models to evaluate the efficacy of this treatment combination.The in vitro studies consistently demonstrated a notable inhibitory effect on the proliferation of various cancer cells when doxorubicin and metformin were administered together. Importantly, the combination regimens exhibited significantly higher inhibition rates and apoptotic indices compared to monotherapy with each individual drug [25], [26], [27], [28], [29]. The study by Ying Li et al. exemplifies the promising efficacy of combining metformin with doxorubicin for breast cancer treatment [30]. Metformin effectively inhibits cellular transformation by suppressing NF-κB activation, leading to reduced cell proliferation through apoptosis induction and cell cycle G0/G1 arrest in MCF7/Adr cells [31]. Moreover, metformin synergistically enhances doxorubicin's antitumor effects in xenograft models and successfully overcomes chemotherapy-induced drug resistance. In vivo experiments further support metformin's potential as a valuable therapeutic option, highlighting its significant antitumor effects. Overall, metformin delays cellular transformation, enhances doxorubicin's efficacy, overcomes drug resistance, and exerts antitumor effects, making it a promising adjunctive therapy in cancer treatment [32].

Another study used mice as experimental models and subjected them to different drug treatments. These included oral administration of metformin at a dose of 3 mg/kg per day and intraperitoneal injection of doxorubicin at a dose of 3 mg/kg per day, twice a week, administered either alone or in combination. Tumor volume was measured and compared among different treatment groups. The results showed that the combined treatment displayed better efficiency in inhibiting tumor growth compared to the administration of each drug alone. Based on these findings, the study concluded that metformin, when used in combination with other chemotherapy agents, has the potential to suppress the growth of lymphoma cells and increase the therapeutic response [32]. In experimental studies, breast cancer cells were subjected to incubation with varying concentrations of DOX, both with and without the presence of metformin. The findings revealed that the combination of metformin with DOX led to a reduction in the IC50 of DOX in MCF-7/DOX cells. Remarkably, this combination mitigated the toxic side effects associated with DOX without compromising its efficacy. Additionally, the combination of metformin with DOX significantly increased cellular apoptosis compared to the administration of DOX alone. The study demonstrated that metformin has the ability to enhance the sensitivity of P-gp overexpressing MCF-7 cells to doxorubicin, a widely used anticancer agent. These results highlight the significance of investigating drug resistance mechanisms in cancer cells, particularly the role of ABC transporters such as P-gp in multidrug resistance (MDR) [25] and specifically examined the effects of combining metformin with doxorubicin on a doxorubicin-resistant thyroid carcinoma cell line was shown. The combination treatment, using doses of 50 and 250 ng/ml, resulted in a further reduction in cell viability compared to treatment with doxorubicin alone. The percentage of viable cells, expressed relative to the control, was significantly lower with the combination treatment than with either drug alone (p < 0.01). These findings suggest that utilizing metformin in combination with chemotherapeutic agents like doxorubicin holds promise as an adjunct therapy for thyroid cancer [33]. To investigate the impact of metformin on cellular transformation and the growth of cancer stem cells, the researchers conducted experiments utilizing breast cancer cell lines and a Src-inducible model. Their findings demonstrated that metformin effectively inhibits cellular transformation by impeding the activation of the inflammatory transcription factor NF-κB. Additionally, when metformin was combined with doxorubicin, it exhibited an antitumor effect that was attributed to a reduction in the functionality of the inflammatory feedback loop [31].

Animal studies have provided evidence of the synergistic effects of metformin when combined with doxorubicin. These studies consistently demonstrate that metformin enhances doxorubicin's ability to reduce breast cancer tumor growth in mice, resulting in a notable decrease of over 50 % in both tumor weight and volume [25,34]. Furthermore, the combination of metformin (200 μg/mL) and doxorubicin (4 mg/kg) has exhibited a synergistic effect in preventing the proliferation of breast and prostate cancer cells over 20 days, particularly effective in inhibiting tumor growth in xenograft mouse models [26,31]. Additionally, studies have shown that the combination of metformin and doxorubicin reduces tumor growth and prolongs recurrence in mice by targeting highly tumorigenic (cancer stem cell-like) cells [35]. When administered for 42 days (200 μg/mL metformin + 4 mg/kg doxorubicin) or 27 days (15 mg/kg metformin + 4 mg/kg doxorubicin), the combination synergistically reduced tumor volume, improved survival rates, and increased apoptosis in mouse breast adenocarcinoma by activating AMPK [35,36]. These findings underscore the potential of combining metformin and doxorubicin as a promising therapeutic approach for breast cancer treatment.

In vitro and in vivo investigations align with findings from a human study. Phase I window clinical trials demonstrate that patients receiving a daily oral dose of 1 g metformin alongside vincristine, dexamethasone, PEG-asparaginase, and doxorubicin exhibited higher response rates, longer median treatment tolerance, complete recovery, and improved relative response compared to those receiving doxorubicin alone for relapsed refractory acute lymphoblastic leukemia [37].

However, contrasting results were observed when combining 2 g/day metformin with 60 mg/m2 non-pegylated liposomal doxorubicin for non-diabetic women with HER2-negative metastatic breast cancer (MBC) in the MYME trial. This trial did not support the anticancer activity of metformin in combination with first-line doxorubicin in MBC. Interestingly, insulin-resistant patients (HOMA ≥ 2.5) experienced a significantly shorter progression-free survival (PFS) [38]. These findings suggest that while both drugs individually inhibit cancer cell viability, the combination regimens with metformin may suppress doxorubicin's cytotoxicity. Additionally, the combination treatment at the highest tolerated dose did not improve PFS or overall survival (OS) [38]. These results highlight the importance of considering context and patient characteristics when evaluating the efficacy of combining metformin and doxorubicin for HER2-negative MBC treatment.

Furthermore, a study by Meyer et al. on BALB/c fibroblasts found that the combination of metformin and doxorubicin exhibited a more potent cytotoxic effect when administered individually compared to co-treatment. These findings raise concerns regarding the concurrent use of metformin during chemotherapy, suggesting careful consideration of the optimal timing and sequencing of these treatments [39].

Discussion

The systematic review provides robust evidence supporting the therapeutic benefits of combining doxorubicin and metformin regimens in cancer treatment. Most in vitro studies consistently demonstrated a significant inhibitory effect of the combined regimen on cancer cell proliferation, resulting in inhibited growth, cell invasion, colony formation, migration, and induction of cell cycle arrest, cytotoxicity, and apoptosis compared to their individual use [25,27,42]. Animal studies further corroborate the synergistic effects of combining metformin with doxorubicin, clearly indicating a remarkable decrease in tumor weight, volume, and growth, as well as an increase in autophagy, remission, survival rate, and apoptosis, leading to prolonged remission compared with the administration of each drug alone [30,40,41]. However, few studies have reported contrasting results by showing an increase in tumor growth when compared with the administration of each drug alone. Metformin has demonstrated a significant impact on treatment tolerability, patient compliance, and the adverse effects of doxorubicin [38].

Synergistic anticancer effects of metformin and doxorubicin can be attributed to their impact on various signaling pathways, including EGFR, AMPK, mTOR, and Wnt/β-catenin [19,21,23]. Accordingly, multiple studies consistently reported the activation of the AMPK signaling pathway as a result of metformin's direct inhibition of the mitochondrial ETC I complex [27,32,33,37,38]. Doxorubicin, through the induction of cellular stress and DNA damage, also activates this pathway. Metformin's dependent effects on the mTOR signaling pathway occur through suppression of mTOR by the activation of the AMPK pathway [23]. Meanwhile, metformin exerts independent inhibition of the mTOR pathway either by inactivating Rag GTPases or upregulating REDD1, a negative regulator of mTOR [52,53]. Doxorubicin has also demonstrated a modulatory effect on the mTOR pathway. There are suggestions that the addition of rapamycin to doxorubicin treatment enhances the antitumor effects by downregulating mTOR activity [54,55], a mechanism similar to the potential effects of metformin as hypothesized and observed in other studies.

Additionally, based on Salim et al.'s study, both metformin and doxorubicin contribute to the reduction of mRNA expression of ERα and EGFR, leading to a potentiated suppressive effect [41,47]. This phenomenon can be attributed to the possible downstream disruptive impact of doxorubicin on signaling molecules like MAPK and PI3K/Akt, as well as the downregulating effect of metformin on the PI3K/Akt pathway, a potent strategy proposed to enhance the cytotoxic effects of doxorubicin [54]. The activation of the MAPK pathway is widely recognized as a significant contributor to doxorubicin resistance [56]. Theoretically, the inhibitory impact of metformin on the PI3K/Akt pathway holds promise for counteracting resistance [57]. Metformin achieves this by activating AMPK, phosphorylating IRS1, and inhibiting the mTOR pathway [57]. This study also reports a reduction in mRNA expression of ERα, which can be attributed to the inhibitory effect of metformin on epoxyeicosatrienoic acid (EET) biosynthesis and subsequent reduction in ERα expression [58].

The potentiated suppressive effect of the metformin and doxorubicin regimen on the Wnt/β-catenin signaling pathway, as reported by Banerjee et al. [34], can also be attributed to the effects of both drugs. Metformin can promote β-catenin stability and accumulation by inactivating GSK3-β, interfering with DVL3-mediated Wnt/β-catenin signaling, promoting β-catenin phosphorylation, decreasing the expression of HNF4α, WNT5A, miRNA-21, and increasing the expression of DDIT3, Klotho, and miR-145 [59,60]. Correspondingly, doxorubicin can suppress the Wnt/β-catenin pathway by suppressing β-catenin activity through downregulating its transcription, facilitating its ubiquitin-proteasome-mediated degradation, and interacting with Dvl-3 [10]. Numerous studies have reported that adding Wnt inhibitors to doxorubicin chemotherapy significantly enhances the anticancer activity of doxorubicin [61,62].

The influence of metformin and doxorubicin on vital cellular processes such as cell death, cell cycle, and autophagy plays a pivotal role in their combined potent effect against cancer cells. Several studies (shown in Table 2) have demonstrated an augmentation in apoptosis induction and cell cycle arrest, specifically involving upregulation of p21 expression and cell cycle arrest at the G1/S transition [40,42,46]. Both doxorubicin and metformin can activate TP53 through AMPK activation. This activation occurs through their binding to p53RE in the promoter region of the P21 gene, consequently enhancing its transcription, leading to cell cycle arrest, autophagy, and apoptosis in cancer cells [63].

Metformin has been reported to prevent G0/G1 transition by decreasing the expression of cyclin D1 [64,65]. Doxorubicin, on the other hand, can induce cell-cycle arrest by preventing the dephosphorylation of CDK-cyclin complexes by phosphorylating CHK1 and CHK2 through amplifying the activity of ATR and ATM kinases [9].

The precise impact of autophagy on cancer therapy remains a subject of debate [66]. Autophagy can act as a tumor suppressor by safeguarding against DNA damage and maintaining genomic stability, and it can impede tumor growth by inducing cell death. However, in certain types of cancer cells, autophagy can paradoxically promote tumor growth by providing nutrients, aiding metastasis, and contributing to treatment resistance in harsh microenvironments [66]. Therefore, the relationship between metformin's ability to upregulate autophagy, due to its activation of AMPK and inhibition of mTOR signaling pathways, and its influence on cancer progression is still a topic of debate, necessitating further investigation.

A study conducted by Barredo et al. reported that the combination of metformin and doxorubicin has the potential to enhance apoptosis dependent on ER stress [37]. Doxorubicin has been found to increase the expression levels of ER stress proteins, including PERK, ATF-6, and CHOP [9]. Conversely, metformin can decrease the expression of GRP78 and activate the UPR-mediated apoptotic pathway through an AMPK-dependent mechanism in ALL cells [67]. Metformin may also modulate the UPR by activating the PERK/ATF4/CHOP axis and inhibiting the ATF6/GRP78 axis. The metformin and doxorubicin regimen increased the susceptibility of ALL cells to ER stress by inhibiting the UPR [37]. In a study by liu et al., it was deduced that metformin can hinder the expression of inflammation-related factors by inhibiting the expression of NLRP3 inflammasome and thus reduce the damage of the inflammatory microenvironment to normal cells, and delay the progression of colorectal cancer [68]. Reports considering the effect of metformin on cellular stress response pathways have produced conflicting results; some studies suggesting inductive effects on the UPR pathway and others indicating suppressive effects. Further investigation is required in this field to gain a comprehensive understanding of the underlying factors of this effect.

Suppression of SphK1 activity, an enzyme that converts sphingosine to the signaling mediator S1P, which promotes cell growth, proliferation, and migration [69], [70], [71]. Correspondingly, Kabel et al. (Table 2) reported that metformin reduces SphK1 activity and shifts the sphingolipid rheostat towards reduced S1P production [36]. Additionally, a study reported that metformin can exert a cytoprotective effect by decreasing the production of ROS [49]. Doxorubicin can initiate ROS production by binding to cardiolipin and triggering electron leakage by disrupting its function; ROS itself can prompt cell cycle arrest and cell death [9].

The effect of metformin on ROS appears to be complex and context-dependent, acting as both an antioxidant and a pro-oxidant. It can exert beneficial effects in some instances while potentially contributing to oxidative stress in others. Thus, despite favorable initial results, further investigation is required to fully understand metformin's effect. These protective effects can be studied in clinical trials to investigate the effects of metformin on doxorubicin-induced toxicities, including cardiotoxicity, bone marrow suppression, nausea and vomiting, alopecia, and neurotoxicity.Accordingly, Nanni et al. reported that adding metformin to chemotherapy regimens decreased the incidence of severe neutropenia in a phase II randomized clinical trial on HER-2-negative metastatic breast cancer patients [38]. Additionally, Chen et al. reported metformin's modulatory effects on doxorubicin-induced cardiotoxicity [72].

The development of drug resistance in cancer therapy is a major challenge, influenced by factors like drug translocation and cancer stem cells, as well as adaptive response mechanisms. P-glycoprotein (P-gp) and ABC transporters can translocate and efflux doxorubicin from cancer cells and cancer stem cells, a mechanism used by cancer cells to gain resistance against cytotoxic agents [73]. The metformin and doxorubicin regimen can prevent cancer cells from gaining multi-drug resistance by inhibiting these transporters [30,46,47]. Another significant reported finding is the selective eradication of cancer stem cells achieved through the metformin-doxorubicin combination. [31,35,74]. CSCs possess intrinsic abilities to resist traditional therapy, such as activation of drug-efflux processes by the ATP binding cassette (ABC) transporters, dormant state transition, and rapid DNA-repair mechanisms [75]. Overexpression of ABC transporters, such as BCRP/ABCG2 and P-gp/ABCB1, are associated with multidrug resistance, including resistance to doxorubicin which can be counterbalanced by metformin's inhibitory effect on theses transporter's activity and expression [76]. Over the past few years, immunotherapy, especially NK cell and T-cell therapy, has exhibited potential in eliminating CSCs [75]. Metformin's boosting effect on NK cells’ cytotoxicity cells can be exploited in this expertise, as well [77]. Additionally, cancer stem cells’ stemness and tumorigenicity are contemplated to be strictly controlled by interactions of multiple signaling networks, including the Hh, Notch and Wnt pathways, and biomolecules, such as cytokines, within the tumor microenvironment [75]. Metformin's selective effect (either alone or in combination with doxorubicin) on these pathways, can have an important role in metformin's selective inhibitory effect on CSCs and combating drug-resistant tumor cells. Moreover, metformin's inhibitory effect on surviving and suppression of STAT3 is completely silimar to the that of LFS-1107 which is introduced as selective inhibitor of BCSCs [78]. Multiple studies have suggested suppressing EMT can increase doxorubicin's sensitivity to doxorubicin resistant cancer cells [9]. Considering phenotype plasticity model, metformin's inhibitory effect on the EMT hinders the conversion between differentiated tumor cells and CSCs [75]. Metformin has shown inhibitory effects on cancer invasion and metastasis by suppressing epithelial-to-mesenchymal transition (EMT). It achieves this by increasing the expression of E-cadherin, a protein that promotes cell adhesion, while decreasing vimentin levels, a protein associated with cell migration [42,[79], [80], [81], [82]]. Metformin achieves this effect by blocking COX-2, an enzyme involved in inflammation, and its consequent PGE2 production, reducing Snail protein expression, IL-6 levels, and inhibiting Bmi-1, a protein involved in self-renewal [42,[83], [84]]. Accordingly, Qu et al. (Table 2) reported that metformin's inhibitory effect on EMT can be used to overcome and reverse doxorubicin resistance [42].

Hirsch et al. (Table 2) reported that the combined regimen of metformin and doxorubicin preferentially inhibits the NF-κB pathway (a key regulator of immunity) in cancer stem cells CSCs compared to non-cancer stem cells (NSCCs) through IkB phosphorylation. They also found that the best timing for administering metformin is near the time of the initial inflammatory stimulus caused by doxorubicin [26]. Downregulation of NF-κB inhibits a wide range of inflammatory cytokines and chemokines, including TNF-α and IL-6 [22]. Metformin can also suppress the phosphorylation of STAT3, a protein involved in cell growth and survival, in monocytes and their differentiation into macrophages [20].

Another important effect of metformin is its impact on the tumor microenvironment (TME), where cancer cells suppress the release of death signals and increase the secretion of growth factors to promote their survival [41]. Metformin's suppressive effect on the PI3K/Akt/PTEN pathway can hinder the cellular effects of growth factors like EGF and TGF-β, suppress their release, and induce the expression of MHC-I on cancer cells, making them more recognizable by the immune system [20]. Additionally, metformin can help prevent T cell depletion and increase the number of CD8+ tumor-infiltrating lymphocytes (TILs) by altering the function of CD8+ T cells and protecting them from cell death and exhaustion. Furthermore, metformin can reduce PD-L1 expression in tumor cells through multiple pathways, leading to enhanced cytotoxic T lymphocyte (CTL) activity and potentiating doxorubicin's performance within the tumor microenvironment [84,85].Metformin's influence on T-cells can be exploited in T-cell based immunotherapies against CSCs [75]. metformin also demonstrates inhibitory properties against angiogenesis and tumor migration. It achieves this by downregulating the expression of vascular endothelial growth factor (VEGF), suppressing HIF-1α-induced expression of factors associated with angiogenesis, and reducing the signaling of platelet-derived growth factor-B (PDGF-B) and platelet-derived growth factor receptor-beta (PDGF-Rβ) [74].

Conclusion

The therapeutic utilization of doxorubicin, despite its pleiotropic effects on multiple cellular pathways, encounters various biological obstacles such as epithelial-mesenchymal transition (EMT), drug resistance, significant adverse effects, and excessive secretion of growth stimulatory factors by immune cells. Concurrent administration of metformin with doxorubicin, due to its effect on overlapping pathways as doxorubicin, provides the opportunity to potentiate doxorubicin's cytotoxic effects not only augmenting doxorubicin's cytotoxic effects but also mitigating the challenges mentioned before. Although substantial evidence supports the concurrent administration of metformin and doxorubicin, further investigations are warranted to elucidate their combined effects, particularly focusing on endoplasmic reticulum (ER) stress and autophagy. It is recommended to direct efforts towards samples exhibiting unfavorable combinatorial effects, with a primary emphasis on unraveling the underlying mechanisms and a secondary focus on identifying negative biomarkers that can be useful in future personalized medicine therapies.

Ethics and integrity policies

Not Applicable.

Funding statement

Not Applicable.

Ethics approval statement

Not Applicable.

Patient consent statement

Not Applicable.

Permission to reproduce material from other sources

Not Applicable.

Consent for publication

Not applicable.

CRediT authorship contribution statement

Fereshtehsadat Jalali: Writing – original draft, Methodology, Data curation. Fatemeh Fakhari: Writing – original draft, Methodology, Data curation. Afrah Sepehr: Writing – original draft, Software, Resources, Data curation. Jaber Zafari: Validation, Software, Project administration, Investigation, Data curation. Behnam Omidi Sarajar: Validation, Methodology, Formal analysis, Data curation. Pouria Sarihi: Writing – review & editing, Writing – original draft, Visualization, Supervision, Conceptualization. Emad Jafarzadeh: Writing – review & editing, Writing – original draft, Supervision, Conceptualization.

Declaration of competing interest

All authors confirm that they have no conflicts of interest related to the submitted manuscript.