Introduction

Gossypol is a polyphenol compound found in cotton plants (Gossypium sp.) It is a seed pigment with a protective role. It is also known as an oral male contraceptive for treating gynaecological disorders. Numerous studies have shown its anti-tumour, antioxidant, antiviral, antimicrobial, and immunomodulatory activities [28]. Nevertheless, gossypol has limited application in medicine as a potential pharmacological agent, mainly due to the narrow therapeutic range of doses, the risk of permanent irreversible sterility [72], and hypokalaemia. This problem led to numerous studies aimed at reducing the side effects and toxicity of gossypol and identifying and develo** new derivative molecules with reduced side effects and toxicity. The mechanism of anticancer activity of gossypol is the induction of apoptosis through the suppression of anti-apoptotic proteins of the Bcl-2 family [85]. Anticancer activity of gossypol is proven on several different cancer cell lines [49]: human breast cancer cells (MCF-7, MDA-MB-231, MDA-MB-468, ZR-75-1, and T47D), pancreatic cancer cells (BxPC-3 and MIA PaCa-2), human colon cancer cells (COLO 225), human cervical cancer cells (HeLa and SiHa cell lines), non-small cell lung cancer (NSCLC) cell lines (H1975), human lung cancer cell lines (H1299 and H358) and prostate cancer cells. Except in China, where gossypol is available on the drug market as an adjuvant used for tumour treatment [85], in the rest of the world, gossypol is still under clinical trials investigation. This comprehensive study aims to summarise all available data on the biological properties of gossypol, particularly its anticancer activity, together with the mechanism of this activity and an overview of clinical studies with gossypol and its medical use.

Review methodology

Information was gathered from various scientific databases, including the National Center for Biotechnology Information (NCBI), PubMed/MedLine, Google Scholar, Scopus and TRIP databases for this comprehensive review of gossypol and its potential anticancer activity. The selected studies were analysed about the structure and plant sources of gossypol and its derivatives, the medicinal use, the bioavailability and scientific studies that confirmed the anticancer properties of the compound. The following MeSH terms: “Antineoplastic Agents/pharmacology”, “Antineoplastic Agents/therapeutic use”, “Gossypium/chemistry: “Gossypol/analogues & derivatives”, “Gossypol/isolation & purification”, “Gossypol/pharmacology”, “Gossypol/therapeutic use”, “Neoplasms/drug therapy” and other keywords such as gossypol, cottonseeds, plant sources, anticancer properties, bioavailability of gossypol, studies in vitro and in vivo, antitumor action, and immunomodulatory effects have been used for the searching. The taxonomy of plants associated with gossypol was validated according to the World Flora Online [77] and chemical structures according to PubChem [51].

Gossypol: general characterisation

Natural sources of gossypol

Gossypol is a yellow crystalline pigment in the cotton plant seeds (Gossypium sp.) of the family Malvaceae [50]. The genus Gossypium consists of about 50 species, and the most cultivated species are Gossypium hirsutum and Gossypium barbadense. Gossypol is present mainly in the seeds but can also be found in the plant's roots, stems, and leaves. Gossypol is present in free form, and its primary function role is to protect the plant from pests and diseases. It bears noting that genetically modified cotton plants have a lower content of gossypol [62]. Gossypol is a polyphenol and a secondary metabolite detected in cotton plants belonging to the genus Gossypium (family Malvaceae). Its role in plants is crucial for development and self-protection [37]. To improve the water solubility and bioavailability of gossypol, Wang et al. [76] used gossypol-loaded pluronic F127 nanoparticles (GLPFNs), which increased bioavailability several times and exhibited higher organ uptake of the drug compared to using gossypol alone. About gossypol derivatives, it bears noting that apogossypol has a slower clearance rate than gossypol [49] with similar in vitro stability, while apogossypol hexaacetate has no oral bioavailability [26].

Molecular mechanism of antitumor action of gossypol

Gossypol exerts its anticancer effects through a complex interplay of molecular mechanisms, leading to distinct biological consequences like apoptosis, autophagy, inhibition of tumor cell viability, angiogenesis, and immunomodulation; these mechanisms are intricately linked and often result in overlap** effects, contributing to the compound's overall antitumor activity.

Apoptosis induction

Inhibition of anti-apoptotic proteins

The main mechanism of gossypol-anticancer activity is inducing apoptosis through suppressing anti-apoptotic proteins of the Bcl-2 family. This effect results from the inhibitory activity of AT-101, which acts as a mimetic agent to Bcl-2 Homology Domain 3 (BH3), downregulating Bcl-2-related proteins in human cancer cells.

Activation of apoptotic pathways

Also, it has been shown that gossypol may induce apoptosis via caspase-dependent and independent pathways. The caspase-dependent anti-tumour effect of gossypol is led by activation of caspase-3 and caspase-9. Apoptosis induced by independent pathways is made by alternations on the mitochondrial outer membrane permeabilisation [85].

Oxidative stress and mitochondrial dysfunction

Gossypol has been shown to induce also cell apoptosis through oxidative stress (Fig. 4). Gossypol treatment has been demonstrated to induce the production of reactive oxygen species (ROS) in tumour cells [80]. Elevated levels of ROS can trigger oxidative stress, DNA damage, and the activation of apoptotic pathways. In the case of multiple myeloma cells, treatment with 80 μmol/L gossypol resulted in a significant increase in cellular ROS levels, leading to ATP depletion, which induces mitochondrial dysfunction. The impaired function of mitochondria further contributes to the activation of apoptosis [80] (Fig. 4).

Fig. 4
figure 4

Apoptosis induction in cancer cells by gossypol. Gossypol interferes with cellular function by causing mitochondrial dysfunction, which leads to an increase in reactive oxygen species (ROS). This accumulation of ROS results in oxidative stress that damages cellular components, including DNA. Concurrently, gossypol's interaction with mitochondria leads to ATP depletion, crippling the cell’s energy supply and further exacerbating cellular stress. The compound also hinders key survival signals by downregulating Akt, a protein essential for cell survival, and c-Myc, a transcription factor that supports cell growth and proliferation. Additionally, gossypol inhibits the activity of telomerase reverse transcriptase (TERT), an enzyme vital for maintaining telomere length and thereby cell longevity. Together, these actions culminate in the activation of the cell’s apoptotic pathways, leading to programmed cell death. ↑ increase, ↓ decrease, telomerase reverse transcriptase (TERT), Adenosine triphosphate (ATP), Cellular myelocytomatosis oncogene (c-MyC), serine/threonine protein kinase (Akt)

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Epigenetic modulation and DNA damage

Recent studies indicated that gossypol may have epigenetic effects on human cancer cells. DNA damage can trigger apoptosis as a protective mechanism to eliminate cells with excessive genetic alterations [25]. Gossypol targets and damages nuclear DNA by upregulating DNA replication and mismatch proteins, among other effects [57]. Gossypol has been shown to block DNA synthesis in HeLa cells by inhibiting key nuclear enzymes, specifically polymerase alpha and polymerase beta. By inhibiting these enzymes, gossypol interferes with DNA replication, impairing DNA synthesis and potentially causing DNA damage [57].

Telomerase activity modulation

Gossypol has been found to modulate telomerase activity in leukaemia cells. Telomerase is an enzyme that plays a role in maintaining the length of telomeres, which are protective caps at the ends of chromosomes. Dysregulation of telomerase activity is commonly observed in cancer cells. Gossypol can modulate telomerase activity through both transcriptional downregulation and post-translational modification of telomerase reverse transcriptase (TERT). The transcriptional downregulation of TERT involves the inactivation of c-Myc, a transcription factor that regulates TERT expression. Additionally, gossypol can inhibit Akt, a signalling pathway involved in cell survival, leading to post-translational modification and inactivation of TERT. These effects on telomerase activity can ultimately result in the apoptosis of leukaemia cells [46].

Induction of autophagy as a complementary process of apoptosis

Gossypol has been shown to induce autophagy, a cellular process involved in the degradation and recycling of cellular components (Fig. 5). Treatment of colorectal cancer cells with gossypol-induced autophagy and apoptosis [39]. The molecular mechanism is different for both, but eventually, their action is to remove unnecessary cells [40]. During autophagy, one of the well-known enzymes, LC3, is transformed from LC3-I to LC3-II. The treatment with gossypol in colorectal cancer cells increased the LC3-II/LC3-I ratio and induced autophagy [39]. On the other hand, in the same cell type, exposure to 20 and 40 µM gossypol significantly decreased Bcl-2 expression. Consequently, it led to increased expression of Bax, hence the release of Cyt-c and activation of caspase 3, which is the final step of apoptosis [39].

Fig. 5
figure 5

Autophagy induced by gossypol in cancer cells. Gossypol stimulates the conversion of LC3-I to its lipidated form LC3-II, which is a key step in autophagy initiation. LC3-II is associated with the autophagosome membrane. The process begins with the initiation of a phagophore, which expands to engulf cellular components targeted for degradation. The maturation of the phagophore leads to the formation of an autophagosome, which then fuses with a lysosome to form an autolysosome. Within the autolysosome, the encapsulated materials are degraded and recycled, providing the cell with a mechanism to remove damaged organelles and proteins. The action of gossypol in promoting this pathway suggests a potential therapeutic mechanism by which cancer cell survival is reduced through the enhanced turnover of cellular components. LC3-I Microtubule-associated proteins 1A/1B light chain 3B, form I, LC3-II Microtubule-associated proteins 1A/1B light chain 3B, form II, PE Phosphatidylethanolamine

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Figure 5 illustrates the role of gossypol in inducing autophagy within cancer cells, highlighting the conversion of LC3-I to the autophagosome-associated LC3-II through lipidation, and the subsequent steps leading to degradation and recycling of cellular components.

Inhibition of tumor cell viability and signaling pathway modulation

Recently, it has been shown that gossypol may act as an inhibitor of the Nrf2/ARE (nuclear factor erythroid 2–related factor 2/antioxidant-responsive element) signalling pathway in cancer cell lines. Nrf2 is a stress-activated transcription factor that binds to the promoter region of the ARE. This signalling pathway is recognised as a potential target for cancer chemotherapy. However, the over-activation of Nrf2 in cancer cells is also responsible for the chemotherapy resistance [29] in a study by Tang et al. [63], gossypol reduced Nrf2 protein stability, leading to the inhibition of the Nrf2/ARE pathway, resulting in a significant decrease of cell viability in human cancer cells and stimulation of cytotoxicity in chemo-resistant cancer cell lines. In cancer cells, tumour necrosis factor-alpha (TNF-α) can stimulate the expression of intercellular adhesion molecule-1 (ICAM-1) through the activation of nuclear factor-kappa B (NF-κB). ICAM-1 is involved in cell adhesion processes and plays a role in inflammation [11]. Not surprisingly, the contraceptive efficacy of gossypol was reported to be over 99% in several studies [12, 36].

The behavioural study on animal models suggests that rats have an aversion to voluntary ethanol drinking because a metabolic reaction between gossypol and alcohol inhibits hepatic alcohol dehydrogenase. Therefore, it increases the development of condensation products between the biogenic precursor amine and the unreacted aldehyde intermediate(s) to form alkaloid-like compounds (Messiha, 1991).

Toxicity, safety and side effects

Gossypol has toxic properties, and hence, it aids in the protection of cotton plants from several insects and/or pathogens. In this sense, animal feed cotton meals could have toxicity on the long-term feed. Besides, it can be a source of human toxicity directly or through the food chain [38]. Free gossypol may cause anorexia, respiratory distress, impaired weight gain, apathy, immunity impairment, cell and blood vessel damage, and heart failure and may lead to death. The main toxicity is male infertility, which could be irreversible and hypokalaemia [16]. Among the main side effects of gossypol noticed during clinical studies are haemolytic anaemia, diarrhoea, and other gastrointestinal-related symptoms [55]. In this sense, there are several methods for cotton meal physical detoxification, viz., dry heating, immersion, puffing, and separation by centrifugation. Chemical detoxification includes extraction, oxidation by oxidising agents, and alkali immersion. Besides, microbial fermentation could reduce free gossypol toxicity [38]. Moreover, the noticed side effects of gossypol could be managed by decreasing the doses and treating symptoms whenever possible [55]. In this context, the derivatisation of gossypol can lead to better biological potentials alongside a lower toxicity [17, 86].

Limitations and future perspectives

The journey of gossypol in medical applications, especially as an anticancer agent, is not without its hurdles. The narrow therapeutic range of gossypol, coupled with significant concerns such as the risk of irreversible sterility and severe side effects like hypokalemia, has restricted its widespread acceptance in clinical settings [3, 72, 83]. These challenges are further compounded by the cytotoxic nature of gossypol and its derivatives, attributed to the phenolic oxygen atoms. Thus, the need to balance efficacy with safety remains a critical area of ongoing research. Despite these limitations, gossypol continues to stand out as a potential natural anticancer agent. Years of research have elucidated its various mechanisms of action, and its efficacy has been demonstrated in numerous in vitro and in vivo studies. The addition of about 25 phase I and II clinical trials and its availability in the Chinese drug market further underscore its therapeutic potential. Addressing the toxicity concerns, there is a growing interest in develo** new gossypol derivatives with minimal side effects and lower toxicity. Moreover, the potential of gossypol to synergize with standard cancer chemotherapies and radiotherapies opens new avenues for its use as a supportive treatment in oncology. The wide distribution of cotton plants, the ease of gossypol extraction, and its proven efficacy against a diverse array of cancer types position it as a highly promising and accessible natural polyphenol molecule. The next step in this journey involves more extensive clinical trials, preferably with larger patient cohorts, to validate its efficacy and safety at a broader scale.

Conclusion

This paper has assessed gossypol's anticancer properties, highlighting its mechanisms of action, including apoptosis induction, autophagy, angiogenesis inhibition, and potential in immunotherapy. While its transition from a contraceptive to a potential anticancer agent has been notable, the findings underscore the need for cautious optimism. Gossypol’s efficacy, evident in various in vitro and in vivo studies, and its progression through phase I and II clinical trials underscore its potential. However, significant challenges, particularly its narrow therapeutic index and toxicity concerns like irreversible sterility and hypokalemia, are obstacles to its widespread clinical adoption. The exploration of gossypol derivatives offers a promising approach to mitigate these concerns. These derivatives aim to reduce toxicity while maintaining or enhancing anticancer effects. Additionally, the potential synergy of gossypol with standard cancer treatments could broaden its application in oncology. Future research should focus on extensive clinical trials to establish a definitive safety and efficacy profile for gossypol and its derivatives. Investigations into optimized formulations, delivery methods, and combination therapies are essential to fully realize its therapeutic potential. The global availability of cotton plants, as a source of gossypol, further supports its potential as an accessible anticancer agent. In summary, gossypol presents as a compound with significant anticancer potential. However, realizing this potential requires a balanced approach that considers both its promising anticancer properties and the challenges it poses. Continued research is crucial for determining its role in future cancer treatment regimens.