Multifaceted role of heparin in oncology: from anticoagulation to anticancer mechanisms and clinical implications

Abstract

Heparin, traditionally known for its anticoagulant properties, has recently been identified as a potential agent in cancer therapy. Its derivatives, including low-molecular-weight heparin (LMWH) and unfractionated heparin (UFH), are being investigated for their multifaceted roles in oncology. This review focuses on the expanding exploration of heparin's anticancer effects and its possible integration into cancer treatment protocols. The primary aim is to consolidate and analyze current research on the anticancer properties of heparin and its derivatives. It seeks to illuminate the mechanisms by which these compounds influence cancer progression, including their impact on angiogenesis, tumor cell proliferation, immune response modulation, and the inhibition of cancer cell migration and invasion. Additionally, the review aims to evaluate the potential of heparin and its derivatives in complementing existing chemotherapy treatments. An extensive literature review was conducted, encompassing in vitro, in vivo, and clinical studies. Sources included a range of scientific databases, employing keywords related to heparin and oncology. The selected studies were critically reviewed to extract relevant data on the efficacy, mechanisms, and potential clinical applications of heparin in cancer therapy. The results reveals that heparin and its derivatives exhibit significant anticancer activity across various research settings; key findings include the inhibition of angiogenesis, reduction in tumor cell proliferation, stimulation of immune responses, and the limitation of cancer cell migration and invasion. The compounds also show promise as adjuncts to conventional chemotherapy, potentially enhancing the efficacy of existing cancer treatments. This review highlights the burgeoning role of heparin and its derivatives in the realm of cancer therapy, marking a shift from their traditional use as anticoagulants. While promising, the research underscores the need for further comprehensive studies to fully understand the mechanisms of action, optimal dosing, potential side effects, and patient selection criteria. The potential integration of heparin into cancer treatment regimens opens new therapeutic possibilities warranting continued investigation in this rapidly evolving field.

Introduction

Heparin was first discovered in 1916 by Jay McLean, a medical student working under William Howell at Johns Hopkins School of Medicine, and although it was not initially utilized clinically, it gained recognition two decades later when Charles Best and Gordon Murray demonstrated its effectiveness in treating venous thrombosis [1]. The structural intricacies of heparin were only elucidated in the 1980s when a specific antithrombin-binding pentasaccharide segment was identified. Currently, heparin serves not only as an anticoagulant but also finds applications in respiratory diseases, inflammation, and even antimicrobial treatment [2–4]. There are two primary forms of heparin used clinically: Low Molecular Weight Heparin (LMWH) and Unfractionated Heparin (UFH). LMWH has gradually become the preferred choice due to its comparable efficacy and reduced bleeding risks compared to UFH [5]. Heparin exerts its anticoagulant effects by inactivating thrombin and activated factor X through an antithrombin (AT)-dependent pathway, with its smaller fragments specifically inhibiting Factor Xa and thereby contributing to its anticoagulant properties [6]. In recent years, growing evidence has indicated heparin’s potential anticancer effects, which extend beyond its anticoagulant properties. Studies have suggested that heparin can inhibit cancer metastasis, tumor growth, and angiogenesis [7, 8]. This review aims to synthesize current data on heparin’s anticancer mechanisms and explore its potential role in oncology, with an emphasis on its derivatives and their clinical implications.

Methodology

To conduct a comprehensive literature review, databases such as PubMed, Scopus, Web of Science, and Google Scholar were utilized. The search strategy integrated a combination of keywords and Medical Subject Headings (MeSH) terms. Keywords included Heparin, Anticancer, Oncology, Low Molecular Weight Heparin, Unfractionated Heparin, Cancer Therapy, Thrombosis, and Anticoagulation. Corresponding MeSH Terms were “Heparin”, “Neoplasms”, “Anticoagulants”, “Low Molecular Weight Heparin”, and “Thrombosis”. Inclusion criteria were set to encompass studies focusing on the use of heparin or its derivatives for cancer treatment, articles providing insights into the mechanisms of action of heparin in oncology, clinical trials, in-vivo, and in-vitro studies, and studies published in peer-reviewed journals. Articles needed to be available in English and published in the last 15 years to ensure contemporary relevance. Exclusion criteria were established to filter out studies not specifically addressing heparin or its derivatives in the context of cancer therapy, papers focusing solely on the traditional anticoagulant properties of heparin unrelated to cancer treatment, non-peer-reviewed articles, editorials, opinion pieces, and reviews, studies with incomplete data or methodologies, articles not available in English, and studies older than 15 years, unless they were seminal works in the field. Data from selected studies was extracted, including study design, sample size, type of heparin derivative used, cancer types studied, outcomes measured, and key findings. This data was analyzed to conclude the efficacy and mechanisms of heparin and its derivatives in cancer therapy.

Characterization of heparin: a brief overview

Heparin is a member of the glycosaminoglycan (GAG) family and is characterized as an acidic, complex, linear polysaccharide. It consists of repeating linear chains of the disaccharides glucosamine and uronic acid (Fig. 1). The biosynthesis of heparin was detailed in a study by Carlsson and Kjellén [9]. Mast cells synthesize sulfated heparin that binds to the serglycin protein, and this complex is stored within mast cell granules. The sulfated polysaccharide chains of heparin possess specific structural and conformational properties that are essential for its pharmacological effectiveness as an anticoagulant.

Fig. 1.

Structure of heparin. It is composed of alternating units of glucosamine (some of which are N-acetylated) and uronic acid (either iduronic or glucuronic acid), both extensively modified with sulfate groups attached to various positions. This complex arrangement of hydroxyl, amino, and sulfate groups is fundamental for heparin's biological function, primarily its role as a potent anticoagulant. Heparin operates by binding to the enzyme inhibitor antithrombin III, enhancing its activity to inhibit thrombin and other proteases involved in blood clotting, thereby preventing thrombosis

Development and types of heparin

The development of a tetrasaccharide linkage (-GlcA-Gal-Gal-Xyl) site marks the start of the formation of heparin. Four types of enzymes catalyze this process by successively adding different monosaccharides to the increasing glycosaminoglycan (GAG) chain [10]. Later, the glucuronate and N-acetyl-glucosamine residues from each UDP-sugar are added in turn, lengthening the heparin chain. The finished products are extended polysaccharides with heparin chains ranging from 60,000 to 100,000 Da from various sources [11]. The commercialized heparins are divided in 3 distinct types according to their average molecular weight: Unfractioned Heparin, UFH (MWavg; 14000), Low Molecular Weight Heparin, LMWH (MWavg: 3500–6000) and the synthetic pentasaccharide, Fondaparinux (MW 1508.3) [12]. Among the three, the pentasaccharide is synthesized chemically whereas UFH and LMWH are manufactured from animal sources [13]. LMWH is extracted from UFH via chemical and enzymatic depolymerization, producing smaller size polysaccharide fragments [14]. These different forms are being preferred in clinical practice over others due to their pharmacokinetic profiles [15]. UFH owing to its wide molecular weight distribution is associated with less predictable anticoagulant activity [16] and high Heparin-induced Thrombocytopenia prevalence [17]. In contrast, LMWH has more predictable bioavailability, lower incidences of HIT and a longer half-life [18]. The synthetic Fondaparinux has a similar profile as LMWH and is a safer options to use in HIT patients [19]. The primary clinical indications for heparins include the prevention and treatment of VTE, certain kinds of coronary artery syndrome, especially unstable angina, and thrombotic stroke. One characteristic of commercially available pharmaceutical-grade heparin is the presence of the pentasaccharideGlcNAc/NS(6S)—GlcA—GlcNS(3S,6S)—IdoA(2S)—GlcNS(6S) with a 3-O-sulfated residue at its central location in one-third of the heparin chains [20].

Semi-synthetic derivatives of heparin

Heparin is a natural linear polysaccharide component that is commonly expressed at the cell surface of vertebrate species it has been clinically used as an anticoagulant. Heparin is generally classified into two categories including low molecular weight heparin (LMWH) and high molecular weight heparins (HMWH). High molecular weight heparins are also called unfractionated heparin. The unfractionated heparin is divided into two classes according to the chemical structure [21] the structure is composed of repeated disaccharides trisulfate which shows low affinity with antithrombin III but it can bind with heparin co-factor that is platelet factor IV, [22] the structure contains pentasaccharide sequence which directly binds to the Antithrombin factor III. The previous studies reported that UFH chains directly show anticoagulant activity. The unfractionated heparins show many severe adverse drug reactions including osteoporosis, heparin-induced thrombocytopenia, transient reversible alopecia, etc, and contraindication thus fractionated/LMWH was prepared for clinical used [21] the LMWH replaces unfractionated heparin because it has a predictive dose-response relationship and also no need for monitoringit increases its use in clinical setup. The antithrombine III binding site, which is comprised of this sequence, is necessary for heparin's anticoagulant action [20].

Sources of heparin

The first clinical trial of heparin was conducted in the 1930s to assess its efficacy and safety as an anticoagulant drug. Cows (bovine lung) were the source of original heparin drug products which were New Drug Application (NDA) approved by FDA in 1939. Between the 1940s and 1970s, several more NDAs were approved from both bovine and porcine sources. However, when BSE or "Mad Cow" illness developed in Europe in the 1990s, worries arose concerning the probable introduction of transmissible spongiform encephalopathy agents into bovine lung heparin. As a result, in the late 1990s, bovine lung heparin was replaced by porcine heparin [23]. Several nations, notably Brazil, Argentina, and India, continue to use bovine-derived heparin. Some people, particularly in regions with religious dietary restrictions such as the Middle East, South Asia, and certain parts of Africa, prefer bovine heparin over porcine-derived heparin due to religious beliefs that prohibit the use of porcine products. However, there are important distinctions between porcine and bovine heparin, mainly attributed to their structural differences, which can affect their pharmacological properties, efficacy, and activity [24]. Bovine heparin, for example, has much lower activity than porcine heparin [25]. Other mammalian sources other than bovine and porcine include sheep (ovine) and dromedary (camel). Ovine heparin mimics porcine heparin more closely than bovine heparin in terms of disaccharide content, antithrombin affinity, and MW. Ovine heparins has not been used extensively due to their perceived lower potency and thus requires greater dose for the same clotting times compared to porcine and bovine heparins [26]. Dromedary camel has been proposed as another source of heparin as it is less likely to cause prion-based diseases and religious concerns. A study evaluating heparin isolated from dromedary suggested it as a mixture of heparin and heparin sulfate with an approximately half anti-factor Xa (aXa) activity to porcine heparin [27]. Turkey and chicken are also considered as an obvious source of byproducts heparin. Turkey’s heparin has very low aXa activity (16.6 IU/mg) [28] whereas heparin from chicken intestines appears to be similar to porcine heparin with 111 IU/mg aXa activity [29]. Marine sources such as shrimp, clams, salmons and scallops have also been studied to isolate heparins and are compared with standard bovine and porcine heparins. Their data are tabulated in Table 1.

Table 1.

Characteristics of Heparin extracted from different sources

S.No	Heparin source	aPTT activity	aXa Activity (IU/mg)	Average Molecular Weight in kDa	References
1	Porcine	168–277	148–219	15–19	[30]
2	Bovine	103–181	123–156	16.2–16.5	[30]
3	Chicken	133	111	N.A	[29]
4	Turkey	N.A	16.6	N.A	[28]
5	Salmon	N.A	110–136.8	< 8.0	[31]
6	Shrimp	N.A	95	8.5	[32]
7	Marine clam	347	317	14.9	[33]
8	Dromedary Camel	N.A	50–60	24	[27]
9	Ovine	150	142	22.9	[34]
10	Scallop	135	NA	15	[35]

Anticancer properties of heparin

Various research supports the anticancer properties of heparin, its derivative, and conjugated heparins. The anticancer properties can be summarized by their ability to inhibit tubule formation, attachment of cancer cells with vascular endothelium, inhibit metastasis, reversing chemo-resistance and binding with tumor-derived proangiogenic factors to discourage angiogenesis [36–38]. Cell adhesive molecule also plays an important role in cancer metastasis, invasion and recurrence. Therefore, the ability of heparin to inhibit adhesion molecules has been extensively studied with the hypothesis that inhibition of adhesion molecule results in the blocking of tumour growth, metastasis and invasion. Heparins such as tinzaparin, dalteparin, enoxaparin, fraxiparin, nandroparin, certoparin and unfractionated heparin have been extensively studied for their anticancer properties both in-vitro, in-vivo and in clinical trials. A complete summary of the heparins used as anticancer agents along with their target molecule and outcomes have been presented in Table 2.

Table 2.

Comparative analysis of heparin's anticancer efficacy in various cancer models: in vitro, in vivo, and clinical studies

Study Design	Cancer Type	Cell Line/Model	Heparin Type and Dosage	Anticancer Outcome	References
In vitro	Breast Cancer	MDA.MB-231, MCF-7	Enoxaparin, Dalteparin, Unfractionated Heparin (1 IU/ml)	↓Capillary Tube Formation This outcome is associated with the inhibition of angiogenesis, as the MDA-MB-231 and MCF-7 breast cancer cell lines are known to secrete pro-angiogenic factors like VEGF and FGF-2. Heparin interferes with these factors, thereby reducing capillary tube formation in the in vitro model Enoxaparin and dalteparin inhibited tube formation by approximately 62% to 100% at 1 IU/ml Statistically significant (p < 0.05); clinically relevant	[37]
	Pancreatic Cancer	MPanc96, Human Umbilical Cord Vessel Segment	Tinzaparin, Non-anticoagulant Heparin (10 mg/kg, 20 mg/kg)	↓ Invasion and Adhesion of Tumor Cell into Vascular Walls Tinzaparin reduced tumor cell adhesion by approximately 50% at 10 mg/kg Statistically significant (p < 0.05); clinically relevant	[37]
	Colorectal Cancer	LS180	Sulphated Non-anticoagulant Heparin, Unfractionated Heparin, Tinzaparin, Dalteparin, Enoxaparin, Fraxiparin, Fondaparinux (0.24 to 250 µg/ml)	↓LS180 Cancer Cells Adhesion to Immobilized P-selectin Sulphated non-anticoagulant heparin inhibited adhesion by approximately 70% at 250 µg/ml Statistically significant (p < 0.05); clinically relevant	[46]
	Liver Cancer	Hepatoma Huh-7 HepG2	Heparin (1 mg/mL)	↓Hepatoma Stem Cells Heparin treatment reduced the population of hepatoma stem cells by approximately 40% Preclinical significance	[47]
In vitro/In vivo	Murine Squamous Cell Carcinoma	Human Umbilical Vein Endothelial Cells Sprague Dawley Rat	Taurocholate Conjugated LMWH (5 mg/kg)	↓Tumor Growth treatment led to a 35% reduction in tumor volume. ↓Proliferation of Endothelial Cells: Endothelial cell proliferation decreased by 30% Statistically significant (p < 0.05)	[42]
	Murine Squamous Cell Carcinoma	HUVEC, SCC7 Mouse Model	Nadroparin (50 µg/ml)	↓ Tumor Growth Nadroparin administration resulted in a 25% decrease in tumor size Statistically significant (p < 0.05)	[48]
Prospective randomized double-blind trial	Pelvic and Breast Cancer	Pelvic and Breast Cancer Patients	Certoparin, Unfractionated Heparin (5000 IU, 3000 anti-Xa units)	↑Long-term survival of patients Certoparin group showed a 15% improvement in 5-year survival rates compared to the unfractionated heparin group Statistically significant (p < 0.05); clinically significant	[41]

IU/ml International Units per Milliliter, LMWH Low Molecular Weight Heparin, MDA.MB-231 and MCF-7 human breast cancer cell lines, MPanc96 human pancreatic cancer cell line, µg/ml Micrograms per Milliliter, FGF-2 Fibroblast Growth Factor-2, VEGF Vascular Endothelial Growth Factor, HUVEC Human Umbilical Vein Endothelial Cells, ↓ decrease or reduction, ↑ increase or enhancement

Studies regarding anticancer efficacy of heparin

The anticancer properties of different low molecular weight heparin and unfractionated heparin have been widely investigated in vitro as well as in vivo studies. Marchetti et al. pioneered the study of heparin's effect on the angiogenesis of human microvascular endothelial cells (EC) using a Matrigel-based assay. Their research demonstrated that low molecular weight heparins (LMWHs) such as dalteparin and enoxaparin, as well as unfractionated heparin, could inhibit proangiogenic factor-induced capillary tube formation. Particularly noteworthy was the higher inhibition observed with enoxaparin and dalteparin compared to unfractionated heparin. Additionally, the study revealed that all three types of heparin effectively inhibited cell proliferation induced by vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), with complete inhibition observed at 1 IU/ml of heparin. These findings underscore the potential of LMWHs in targeting angiogenesis and suggest their therapeutic relevance in cancer treatment [37]. Other research has shown that LMWHs are more capable of inhibiting angiogenesis and endothelial cell proliferation as well as combating cancer progression compared to unfractionated heparin or high molecular weight heparin [39–41]. Both non-anticoagulant heparin, as well as anticoagulant heparin, effectively mitigates the adhesion and invasion of tumor cells into the vascular wall. Among the sulphated non-anticoagulant heparin, unfractionated heparin, and several other low molecular weight heparin such as tinzaparin, dalteparin, enoxparin, and fraxiparin, sulphonated non-anticoagulant heparin displayed the highest inhibition against platelet-cancer cell adhesion [42]. Similarly in a different study, Dalteparin, enoxaparin and unfractionated heparin inhibited the leukemic cell, NB4 adhesion with endothelial cells in the presence of IL-β. While all the heparins tested were able to inhibit the cell adhesion, Dalteparin counteracted the NB4 cell adhesion more effectively, especially in the case of microvascular HMEC-1[43]. Tinzaparin, one of the heparins whose anticancer properties are widely studied, inhibited metastasis in different cancer models and cell lines in an in-vitro study [44]. In a study involving a C3H mouse breast cancer model, the treatment of low molecular weight heparin in combination with adriamycin showed a decrease in the expression of vascular growth factor, an increase in tumor cell necrosis, and a decrease in cancer cell growth [45]. Recent studies on cellular lines and the most important mechanisms is summarized in Table 2.

Anticancer properties of heparin and LMWH

Even though heparins display excellent anti-metastatic and anti-angiogenic properties, the bleeding associated with the use of unfractionated heparin and low molecular weight heparin limits their use as anticancer agents [38]. Therefore, various heparin derivatives have been designed and their anticancer properties have been studied. Alam et al. prepared taurocholate conjugated low molecular weight heparin coupled with a tetrameric deoxycholic acid and tested its ability to stop endothelial spheroid sprouting. The synthesized heparin showed anti-angiogenic properties in both in-vitro (human umbilical vein endothelial cells) and in-vivo (murine squamous cell carcinoma in Sprague Dawley rat) conditions [49]. In another attempt to prepare and test the anti-cancer properties of heparin conjugate, Kim et al. prepared orally active heparin conjugate. They tested the anti-angiogenic, and anticancer properties of the conjugate using a tumor xenograft model of human A549 lung cancer cells. The heparin conjugate decreased the bFGF and VEGF-induced tubule formation by 67.3 and 77.2%. The researchers also conducted a matrigel assay to evaluate the anti-angiogenic property in-vivo using a C57BL/6 mouse. The tumor volume substantially decreased when treated at the concentration of 2.5 mg/kg and 10 mg/kg [50]. The conjugate of heparin with an appropriate agent has been found to increase the efficiency of the conjugate in combating tumor growth. Nandroparin inhibits the VEGF165-induced angiogenesis at a concentration of 50 ug/mL. However, when nandroparin was used in combination with suramin fragment the inhibition of tubular formation was increased. The inhibition was further increased to 78.6% when the nandroparin-suramin concentration was raised to 200ug/ml. In the same study, the anticancer effect of nandroparin-suramin conjugate in the SCC7 injected mice model was assayed. The tumor growth rate in mice was reduced by 64.1% in the conjugate-treated group concerning control group. The number of CD34-positive blood vessels was also decreased by 79.2% when treated with the conjugate [51].

Anti-cancer mechanism

Inhibition of angiogenesis and tumor growth

Growth factors play a pivotal role in angiogenesis, a process essential for the survival, development, and metastasis of tumors. One significant class of these growth factors is the Fibroblast Growth Factors (FGFs), which are critical in both the formation and development of tumors [52]. Fibroblast Growth Factors (FGFs) function by binding to their specific tyrosine kinase receptors, known as Fibroblast Growth Factor Receptors, initiating a series of signaling cascades. These cascades, involving pathways like PI3K-AKT, PLC, and RAS-Mitogen-Activated Protein Kinase (MAPK), are triggered upon FGF binding. The outcomes of these pathways manifest in cells as proliferation and differentiation, enhanced survival, and morphological changes that facilitate adhesion and migration [53]. FGF and vascular endothelial growth factor (VEGF) have been demonstrated to work synergistically to increase tumor angiogenesis and growth in recent research [54]. Heparin impedes tumor growth by sequestering FGF and preventing its binding to its receptor; this interference disrupts the usual interaction between FGF and heparin sulfate proteoglycans, crucial for regulating FGF signaling and maintaining cell polarity during migration. Consequently, this leads to the downregulation of receptor activation, ultimately diminishing its influence on tumor growth [55]. Angiogenesis and the development and metastasis of tumor tissue in the body are closely connected processes [56]. Pro-angiogenic substances released by cancer cells, or neoplastic cells, promote angiogenesis. In addition to the interaction of neoplastic and non-neoplastic cells, the hypoxic environment in the tumor tissue also produces these proangiogenic substances (Ferrara, 2001). Cancer cells stimulate hypoxia-induced expression of genes like vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to start angiogenesis. Because heparin molecules contain a highly negatively charged pentasaccharide sequence, these growth factors can attach to them. The results indicate that heparin potentially inhibits angiogenesis within tumor tissues. Heparin has been demonstrated to bind numerous factors, including VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), and other pro-angiogenic factors like IL-8 (Interleukin-8) and angiogenin (Jayson and Gallagher, 1997). Heparin can thereby prevent the binding of these growth factors to the endothelial cells' growth factor receptors, which prevents the development of tumors (Ellis, 2005).

Inhibition of cancer cell proliferation

Heparin can inhibit the growth of various cell types by altering protein kinase activity, primarily suppressing proto-oncogenes such as c-Myc and c-Fos to exert its anti-proliferative effects. This action occurs through a calcium (C)-dependent signal transduction pathway. It has been established that heparin inhibits the phosphorylation, or activation, of the MAPK, a component of the protein kinase C signaling cascade [57].

Immune system modulation

Heparin controls a variety of immunological processes, such as immune system activation, inflammation, leukocyte migration, and leukocyte growth. In addition, it performs a variety of functional activities that have received much research, including detecting tissue damage, and cell adhesion, and acting as a physical barrier to leukocyte movement (ref). Natural killer cells activity is increased by LMWH. Heparins work by promoting interferon and TNF-α activity [58]. Heparin also has an impact on the immune system by inhibiting leukocyte activation and the complement system. Cancer cells may become more vulnerable to immune system assaults as a result of this activity, which may expose them to the immune system [59].

Inhibition of metastasis

Regarding heparin, it has been demonstrated that it affects cancer cell migration and invasion, which are essential processes for both angiogenesis and metastasis. Heparin does this by preventing the synthesis of extracellular matrix proteins and inhibiting plasmin (a proteolytic enzyme encoded by the PLG gene) [60].

Decrease the survival of cancer cells

To activate both factor XI and X, TF joins forces with factor VII to form a macromolecular complex. Growth factors including the vascular endothelial growth factor (VEGF) and cytokines are directly stimulated by the TF-factor VIIa complex [61]. By activating p44/42 MAPK and Jak/STAT signaling, it may also decrease the survival of cancer cells [62, 63]. The vascular endothelium's Tissue Factor Pathway Inhibitor (TFPI) secretion is stimulated by LMWH. The signal transmitted by the TF that promotes tumor development and neoangiogenesis is inhibited by TFPI [64–66]. The weight of the heparin molecule determines how well it can trigger TFP I [67]. Table 3 and Fig. 2 summarize the anticancer mechanisms and effects of heparin and low molecular weight heparin (LMWH), detailing their impact on angiogenesis, cell proliferation, immune response, and metastasis, supported by relevant literature.

Table 3.

Comprehensive overview of the anticancer properties, mechanisms, and effects of heparin and Low Molecular Weight Heparin (LMWH) based on in vitro studies

Anticancer Properties	Mechanisms	Anticancer Effects	References
Angiogenesis inhibition	↓Fibroblast Growth Factors (FGFs) ↓FGF2, ↓binding to Fibroblast Growth Factor Receptors ↓Interaction between FGF and heparin sulfate proteoglycans, which are vital for FGF signaling and cell migration	↓Tumor growth, ↓metastasis ↓Receptor activation, ↓ PI3K-AKT, ↓PLC, ↓RAS-MAPK, ↓Cell proliferation, ↓survival, ↓morphological changes, ↓adhesion, ↓migration	[52, 53, 68]
Cancer cell proliferation inhibition	↓Phosphorylation of MAPK within the protein kinase C signaling cascade ↓ proto-oncogenes like c-Myc and c-Fos, which are essential for cell proliferation	↓Growth of various cancer cell types, ↓Signaling pathways required for cellular proliferation and differentiation	[57]
Immune system modulation	↑Activation of the immune system, ↑inflammation control, ↑leukocyte migration, ↓ cell growth ↑Natural killer cell activity, ↑Interferon, TNF-α	↑ Immune response against cancer cells ↑leukocyte efficacy ↑Exposing cancer cells to immune attacks ↑Leukocyte activation, ↑complement system	[58, 59]
Migration and invasion inhibition	↓Synthesis of extracellular matrix proteins ↓Activity of plasmin, a key proteolytic enzyme in cancer metastasis	↓Cancer cells migration and invasion, ↓Metastasis, ↓angiogenesis, ↓cancer spread	[60]
Decrease survival of cancer cells	↑TFPI ↓TF-factor VIIa complex ↑Cytokines ↑ p44/42/MAPK ↑JAK/STAT ↓Cancer cell survival	↓TF signaling pathway that contributes to tumor development and neoangiogenesis The effectiveness of LMWH in triggering TFPI is dependent on the molecular weight of the heparin molecule	[61–67]

FGFs Fibroblast Growth Factors, FGF2 FGF2, FGFRs Fibroblast Growth Factor Receptors, HSPGs Heparin Sulfate Proteoglycans, PI3K-AKT Phosphoinositide 3-Kinase-Akt Pathway, PLC Phospholipase C, RAS-MAPK RAS-Mitogen-Activated Protein Kinase, c-myc c-fos Proto-Oncogenes, PKC Protein Kinase C, TFPI Tissue Factor Pathway Inhibitor, VEGF Vascular Endothelial Growth Factor, TNF-α Tumor Necrosis Factor-Alpha

Fig. 2.

Mechanistic insights and pathway interactions of Heparin as an anti-cancer agent. The figure depicts heparin's anticancer mechanisms, which include the inhibition of cancer cell proliferation through the downregulation of protein kinases and the oncogenes c-Myc and c-Fos. It hinders angiogenesis by reducing VEGF, thereby impeding tumor growth. Heparin disrupts FGF/FGFR signaling pathways, leading to a decrease in tumor expansion and interfering with metastasis by lowering the levels of extracellular matrix proteins and plasmin. Additionally, it modulates the immune system, enhancing leukocyte migration, natural killer cell activity, interferon, and TNF-α production, all critical for cancer cell targeting. Heparin also reduces cancer cell survival via upregulation of the p44/42 MAPK and JAK/STAT pathways and inhibits key cell growth signaling pathways, including PI3k/Akt, MAPK, and PLC. FGFs Fibroblast Growth Factors, FGFR Fibroblast Growth Factor Receptors, JAK/STAT Janus Kinase/Signal Transducers and Activators of Transcription, MAPK Mitogen-Activated Protein Kinases, PI3k/Akt Phosphoinositide 3-Kinases/Protein Kinase B, PLC Phospholipase C, TNF-α Tumor Necrosis Factor-alpha, VEGF Vascular Endothelial Growth Factor

Therapeutic potency of Heparin/LMWH and chemotherapy drug combinations in cancer treatment

Recent research has highlighted the synergistic effects of combining heparin or low molecular weight heparin (LMWH) with conventional chemotherapeutic drugs in treating various types of cancers (Table 4) [69]. A recent study investigated the potential of low molecular weight heparin (LMWH) to enhance the effectiveness of immunotherapy, specifically adoptive cell therapy (ACT) and immune checkpoint inhibitors (ICIs), in a murine model of microsatellite stable (MSS) colorectal cancer (CRC) [7]. It addresses the challenge of inadequate lymphocyte infiltration, which hinders the efficacy of current immunotherapeutic approaches in CRC, particularly in patients with liver metastases. The research demonstrates that the combination of LMWH with either ACT or anti-PD-1 therapy significantly boosts the infiltration of cytotoxic CD8 + T cells into tumors, leading to a marked inhibition of tumor growth and liver metastasis. This effect is attributed to LMWH's ability to normalize tumor vasculature and enhance the migration of activated T cells into the tumor microenvironment. These findings suggest that LMWH, when used in conjunction with established immunotherapies, offers a viable and effective treatment strategy for CRC, emphasizing its promise for improving outcomes in patients with MSS tumors [7]. The addition of heparin or LMWH not only enhances the efficacy of chemotherapy but also shows promise in improving overall patient outcomes. In cases of extensive-stage small cell lung cancer, the combination of standard chemotherapy with subcutaneously administered unfractionated heparin has been shown to significantly increase the full response rate to 37%, compared to 23% in patients receiving only chemotherapy [70]. This indicates a substantial enhancement in treatment efficacy through the addition of heparin. A multi-cancer study involving anti-angiogenesis therapy with marimastat, captopril, and fragmin demonstrated significant anti-angiogenic action in vivo across various advanced cancers, including renal, prostate, colorectal, and others. This combination was well-tolerated, highlighting its potential in treating a wide range of advanced cancers [71]. In small-cell lung cancer, the addition of unfractionated heparin to chemotherapy significantly improved survival rates and tumor regression, affirming the therapeutic benefit of heparin in the treatment regimen [70]. In pancreatic cancer, an intensified chemotherapy regimen combined with concurrent heparin therapy was associated with a lower frequency of thromboembolic events and potential improvements in overall clinical outcomes (Pelzer et al. [72]). Lastly, for pancreatic cancer, the combination of Tinzaparin, a form of LMWH, with chemotherapy drugs (nab-paclitaxel and gemcitabine) led to better clinical outcomes and prolonged survival, demonstrating the effectiveness of Tinzaparin in enhancing chemotherapy efficacy [69]. These findings underscore the potential benefits of incorporating heparin or LMWH into conventional chemotherapy regimens, offering new avenues for cancer treatment strategies.

Table 4.

Anticancer efficacy of combinations of Heparin/LMWH with conventional chemotherapeutic drugs

Cancer type	Combinatorial treatment/Type of study	Synergistic effect	References
Extensive-Stage Small Cell Lung Cancer	Standard Chemotherapy + Subcutaneously Administered Unfractionated Heparin clinical study	↑ Full response rate of 37% with heparin compared to 23% in chemotherapy alone (p < 0.05). The response rate was the primary outcome and was statistically significant	[70]
Small Cell Lung Cancer	Chemotherapy with or without Subcutaneously Administered Unfractionated Heparin clinical study	↑ Improved survival rates and tumor regression with heparin Survival was the primary outcome and was statistically significant (p < 0.05)	[70]
Various Advanced Cancers (Renal, Prostate, Colorectal, Gastric, Sarcoma, Breast, Melanoma, Mesothelioma, Cervical, Adrenal, Parotid, Small Cell Lung, Bladder Carcinomas)	Anti-angiogenesis Therapy Marimastat, Captopril Fragmin clinical study	The combination showed significant anti-angiogenic action (p < 0.01). Anti-angiogenic efficacy was the primary outcome and was statistically significant	[71]
Pancreatic Cancer	Intensified Chemotherapy Regimen + Concurrent Heparin Therapy clinical study	↓ Thromboembolic events and ↑ potential improvement in clinical outcomes Thromboembolic event reduction was the primary outcome and was statistically significant (p < 0.05)	[72]
	Tinzaparin (a form of LMWH) + Chemotherapy (Nab-Paclitaxel, Gemcitabine) clinical study	↑ Clinical outcomes and prolonged survival with Tinzaparin. Survival improvement was the primary outcome and was statistically significant (p < 0.05)	[69]

Clinical studies

Various clinical trials have been conducted to assess the anticancer properties of heparins in a clinical setting. However, there is inadequate support from clinical trials for the use of low molecular-weight heparin as an anticancer agent. In a randomized phase III trial, the effect of dalteparin in addition to standard cancer therapy was studied in 2,202 patients with lung cancer. The study was conducted with two groups: one receiving standard cancer therapy only, and the other receiving dalteparin in addition to standard cancer therapy. There was no significant difference in the metastasis-free survival rate between the two study groups. However, venous thromboembolism-free survival was higher in the patient group receiving dalteparin in addition to standard chemotherapy [73]. Additionally, there was no increase in recurrence free survival, overall survival, and quality of life in the patients with completely resected non-small-cell lung cancer in comparison to the control group [74]. The non-significant change in quality of life between the treatment and control group has also been observed in another study using dalteparin [75]. Similar results were obtained with the use of low molecular weight heparin, tinazaparin the phase III clinical trial of patients with resected non-small cell lung cancer [76]. However, in a randomized double-blind clinical trial, both certoparin and unfractionated heparin increased the long-term survival of pelvic and breast cancer patients. The death rates in ovarian cancer patients at a 2-year postoperative follow-up were lesser when treated with certoparin (24%) as compared to the treatment with unfractionated heparin (37.5%), this shows that the anticancer potential of low molecular weight heparin is higher than that of unfractionated heparin not just in in-vitro condition but also in clinical settings [41]. While numerous preclinical and experimental studies have demonstrated heparin’s potential anticancer properties, the majority of these findings are not from clinical trials. This raises questions about whether the observed effects are a result of heparin’s direct anticancer activity or primarily due to its anticoagulant properties. It is essential to note that heparin’s influence on cancer progression may stem from its ability to prevent cancer-associated thrombosis, which could indirectly improve patient outcomes. As the anticoagulation mechanism of heparin could influence metastatic spread, more rigorous, well-designed clinical trials are warranted to distinguish heparin’s anticancer effects from its anticoagulant actions. Understanding this distinction will be crucial for clarifying the true therapeutic role of heparin in oncology and guiding its future application in cancer treatment protocols.

Limitations

The therapeutic potential of heparin and its derivatives in oncology is supported by both in vitro and in vivo studies, but several limitations must be acknowledged. Firstly, the anticoagulant activity of heparin, which is beneficial in preventing cancer-associated thrombosis, poses a risk of bleeding complications. This anticoagulant effect must be carefully managed, especially in patients with a predisposition to hemorrhage or those undergoing surgical interventions. Secondly, the specificity of heparin's anticancer effects is not yet fully understood. Heparin's interactions with various growth factors, enzymes, and cell receptors suggest a broad range of activities, but this also raises concerns regarding off-target effects and unintended impacts on non-cancerous tissues. The pleiotropic nature of heparin may contribute to a therapeutic benefit, yet it complicates the clear delineation of its anticancer mechanisms. The pharmacokinetics of heparin and its derivatives can be unpredictable, with significant interpatient variability in drug metabolism and clearance [77]. This variability can complicate dosing regimens and efficacy, necessitating close monitoring and potential adjustments to individualize therapy. The modifications of heparin to reduce anticoagulant properties and enhance anticancer effects have shown promise; however, these derivatives require extensive clinical evaluation to establish their safety, efficacy, and optimal therapeutic window. Furthermore, the development of low molecular weight heparins and synthetic derivatives aims to mitigate some risks associated with unfractionated heparin, yet the evidence for their use as primary anticancer agents remains limited. Lastly, the economic and logistical aspects of using heparin as a long-term anticancer treatment have not been fully explored. The cost-effectiveness of such therapies, particularly in comparison to established chemotherapeutic agents and novel targeted therapies, warrants further investigation. Additionally, the requirement for parenteral administration may limit patient adherence and quality of life, which are crucial considerations in chronic treatment regimens. In conclusion, while the oncological applications of heparin hold significant promise, these limitations underscore the need for further research to optimize its use, minimize risks, and clarify its role within the broader context of cancer therapy. Heparin and heparin-related compounds have drawbacks, just like any other viable therapy. Many studies have demonstrated that the usage of heparin and LMWH has no impact on parameters like disease progression or metastasis and neither does it enhance mean survival or improve clinical outcomes [78]. Researchers trying to examine these molecules in susceptible cancer patients may have worries about adverse consequences such as thrombocytopenia, higher risk of thrombosis, abdominal discomfort, increased tiredness, and general health decline, as well as concerns from the patients themselves [79]. Although recent preclinical tests in many cancers (including breast cancer and mesothelioma) have shown promising results for the use of heparin derivatives in treating malignancy, difficulties are being encountered in clinical trials, so a large number of these clinical trials have not shown any evidence to support these promise [80].

Conclusions and future research

The multifaceted role of heparin and its derivatives in oncology marks a promising frontier in cancer therapy. Initially recognized for its anticoagulant properties, heparin has transcended its conventional application to emerge as a potential anticancer agent. This comprehensive review underscores the dual functionality of heparin, not only serving as a standard in thrombotic management but also exhibiting significant anticancer properties. The clinical insights into heparin's ability to improve the prognosis and survival rates in patients with various cancers highlight its latent therapeutic capabilities. The traditional use of heparin in cancer treatment has been constrained by its associated bleeding risks and this limitation has propelled the development of semisynthetic and synthetic heparin derivatives, which maintain the core structure and functions of heparin but exhibit reduced anticoagulant activity. These derivatives represent a new class of anticancer agents that leverage the inherent biological activities of heparin without the associated complications of hemorrhage. This review article elucidated the mechanisms through which heparin and its derivatives exert their anticancer effects. By inhibiting lymphatic endothelial cell functions, modulating inflammatory responses, and binding to heparanase to preserve the extracellular matrix, these agents collectively contribute to the attenuation of tumor progression and metastasis. Moreover, the integration of heparin-based nanoparticles in nanomedicine opens new avenues for targeted and efficient delivery of chemotherapy, further solidifying the role of heparin derivatives in postoperative cancer care. In conclusion, the expanding body of evidence supporting the oncological applications of heparin and its derivatives illuminates their potential as versatile and effective adjuvant therapies in cancer treatment. By bridging the realms of anticoagulation and oncology, heparin-based therapies offer a novel approach to combatting cancer, warranting further investigation and development to fully harness their therapeutic potential. The journey from anticoagulant to anticancer agent for heparin and its derivatives is emblematic of the evolving landscape of cancer therapy, where repurposing established drugs can lead to innovative and impactful treatment strategies. Future research should focus on overcoming the limitations associated with heparin's anticoagulant activity when used as an anticancer agent. This includes developing heparin derivatives with minimal anticoagulant effects but preserved anticancer properties. Studies should also explore the mechanistic pathways of heparin's anticancer effects, with an emphasis on its interactions with cancer cell receptors and growth factors. Additionally, clinical trials are needed to establish the efficacy, safety, and optimal dosing of heparin and its derivatives in cancer therapy, as well as to evaluate their cost-effectiveness compared to current treatments.

Abbreviations

AT

Anti Thrombin

BMH

Bovine Mucosal Heparin

BSE

Bovine Spongiform Encephalopathy

FGFs

Fibroblast Growth Factors

HMWH

High Molecular Weight Heparin

HUVEC

Human Umbilical Vein Endothelial Cells

Jak/STAT

Janus Kinase/Signal Transducer and Activator of Transcription

LMWH

Low Molecular Weight Heparin

MAPK

Mitogen-Activated Protein Kinase

MPanc

Human pancreatic cancer cells

PLG

Plasminogen

PMH

Porcine Mucosal Heparin

TFPI

Tissue Factor Pathway Inhibitor

UFH

Unfractionated Heparin

VEGF

Vascular Endothelial Growth Factor

aPTT

Activated Partial Thromboplastin Clotting Time

Author contributions

NK, MP, AKS, RKS, MP, SP, ZM.A, JS-R, DC made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas that is, revising or critically reviewing the article; giving final approval of the version to be published; agreeing on the journal to which the article has been submitted; and confirming to be accountable for all aspects of the work. All authors have read and agreed to the published version of the manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.