Approximately 90% of cases of CML in patients are caused by the reciprocal translocation of chromosome 9 and chromosome 22 t(9;22)(q34;q11) that produces a chimeric Bcr-Abl gene (46). Patients with CML are generally treated with the first-generation tyrosine kinase inhibitor imatinib mesylate (IM). However, only 10% of the recipients showed positive responses, while others were resistant to IM treatment (47). Thus, efforts have been made to identify potential alternative and/or combined treatments for CML.

A study of imatinib-resistant CML cells (48) suggested that HC sensitised the cells to apoptosis via the induction of tumour-necrosis-factor-related-apoptosis-inducing ligand (TRAIL) mediated by ROS homeostasis. In the study, imatinib-resistant K562 cells were treated with 4 µM of HC alone or combined with 200 ng/ml TRAIL. Treatment with HC or TRAIL alone for 24 h induced only 5–10% cell death, while combined HC and TRAIL treatment for 24 h induced a significant cytotoxic effect with an increase in apoptosis of up to 81% (48). ROS played a primary role in the HC-mediated sensitisation of the cells to TRAIL, probably acting via the anti-apoptotic proteins X-linked inhibitor of apoptosis protein and FLICE-inhibitory protein. This was evident by the dose-dependent increase in ROS levels when the imatinib-resistant K562 cells were treated with HC and TRAIL together compared with the single HC treatment. However, ROS production was not affected by TRAIL (48).

Another study found that HC induced ROS in a time-dependent manner, resulting in ROS build-up and a state of high oxidative stress, which subsequently triggered phosphorylation of the c-Jun N-terminal kinase (JNK) pathway. The activation of JNK signalling by HC was suggested to induce the phosphorylation of endothelial nitric oxide synthase (eNOS), resulting in the generation of nitric oxide (NO) and, ultimately, cell death (25). However, HC alone did not increase extracellular signal-regulated kinase (ERK) levels or the activation of p38 in K562 cells, suggesting that the anticancer effect of HC is mediated by a mitochondrial ROS-dependent eNOS-mediated route (25). However, in another study, the activation of JNK with a combination of HC and buthionine sulfoximine (BSO) for 24 h stimulated the ERK pathway (49). Furthermore, the intracellular glutathione (GSH) level of the K562 cells was not affected by the HC treatment (25).

HC exhibited potent anti-CML effects in an in vivo nude mouse model with xenografts formed from leukaemia cell lines expressing wild-type and mutant Bcr-Abl. The results showed that the oral administration of 100 mg/kg HC twice daily for 5–7 days reduced subcutaneous tumour growth in the xenograft-bearing animals (25). Furthermore, HC was able to kill CML cell lines harbouring mutant T315I, one of the most common mutations causing imatinib resistance in patients, via the activation of JNK, leading to increased NO production via the phosphorylation of eNOS.

There is evidence that HC interacts synergistically with other treatments; for example, a study showed that 100 µM BSO combined with 10 µM HC potentiated CML cell death by disrupting the redox equilibrium of cells via the reduction of intracellular GSH and promotion of ROS generation. Furthermore, the combination of BSO and HC acted via the GSH-ROS-JNK-ERK-iNOS pathway and the partial activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins (49). Reactive nitrogen species produced by iNOS overexpression may also be involved in the induction of apoptosis in CML (49). This combined treatment might improve upon the efficacy of standard chemotherapeutics in halting the progression of CML cells.

In another study, a combination of 5 µM HC with 5 µM curcumin induced the accumulation of superoxide and H₂O₂, activated JNK and p38 in the mitogen-activated protein kinase (MAPK) cascade and induced the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream mediators S6 kinase and eukaryotic translation initiation factor 4E binding protein 1, causing apoptosis in K562 cells (50). Although it was suggested that ER stress might induce apoptosis via activation of the mTOR signalling pathway, the underlying mechanism remained unclear. When used singly, these doses of HC and curcumin did not induce apoptotic changes in the CML cells. Also, treatment of the cells with apoptosis-mediating concentrations of HC alone inhibited mTOR signalling, while the combined treatment with HC and curcumin activated both mTOR and MAPK pathways; these effects were dependent on the generation of ROS and led to the apoptosis of CML cells (50).

Breast cancer is the most frequently diagnosed cancer worldwide, with an estimated number of cases of over two million in females in 2020 (51). Breast cancers are classified into several molecular subtypes based on various characteristics, including their histology, metastatic potential, mutations, disease progression and response to therapy (52). Numerous chemotherapies inhibit the development of breast cancer by preventing DNA damage (53) or by blocking oestrogen activity, with examples including tamoxifen (54) raloxifene (55), aromatase inhibitors (56), polymerase inhibitors (57), human epidermal growth factor receptor 2 inhibitors and trastuzumab (58). Unfortunately, due to the severe side effects or chemoresistance that they induce, some of these treatments do not improve patients' morbidity or mortality (59).

A recent study indicated that HC acted as an antioxidant on Ehrlich ascites carcinoma (EAC) cells in an in vivo experiment using Swiss albino mice. The EAC-inoculated mice exhibited a high malondialdehyde level, indicating elevated ROS generation (60). The study showed that when administered orally for 21 days at doses of 200 and 400 mg/kg body weight, HC effectively reduced the volume of the EAC tumours in mice, indicating its anticancer potential. In addition, as the level of oxidative stress was significantly reduced, the lifespan of the tumour-bearing mice was increased (60).

Pancreatic cancer is the 14th most common cancer and the 7th most frequent cause of cancer-associated death worldwide (61). The standard treatment for advanced pancreatic cancer is gemcitabine (62). However, adjuvant therapy using a combination of oxaliplatin, irinotecan, leucovorin and fluorouracil, known as FOLFIRINOX, is also used due to its efficacy and ability to promote significantly longer survival than gemcitabine (63). Unfortunately, ~80% of cases of pancreatic cancer progress into metastatic cancers with the development of liver metastases within 2 years of the treatment regime, inclusive of adjuvant therapy (64). Thus, the search for an alternative treatment or adjuvant continues to be crucial.

The proliferation, migration and invasion of pancreatic cancer cells can be inhibited by HC treatment, as is evident by the inhibition of genes associated with epithelial-mesenchymal transition, i.e., goosecoid homeobox and snail family transcriptional repressor 2, and increased DNA damage as revealed by the comet assay (27). The HC concentration used for the migration assay and gene expression analysis was 25 µM for 24 or 48 h. However, higher HC doses were used for protein analysis, i.e., 50 and 100 µM for 48 h. The induction of apoptosis was confirmed by the activation of caspase-3, −8, and −9, the cell cycle proteins cyclin B1 and cell division control 2, cell division cycle 25C and apoptotic-associated proteins, including PARP, BH3 interacting domain death agonist, B-cell lymphoma 2 (Bcl2), Bcl2 associated X and survivin. The induction of apoptosis was suggested to be JNK-dependent. Furthermore, proteins associated with DNA damage, including Ser-139-phosphorylated H2A histone family member X and p53 binding protein 1, and genes DDIT3 and DNA polymerase β were upregulated in MIA PaCa-2 and PANC-1 pancreatic ductal adenocarcinoma (PDAC) cells treated with HC. Notably, HC exhibited higher half-maximal inhibitory concentration (IC₅₀) values, indicating lower toxicity, in a panel of non-cancerous cells comprising L929 and NIH-3T3 mouse fibroblasts and human 293 cells compared with PDAC cells (27).

Aside from active surveillance, which is a viable method of management for localised PCa, the two primary curative forms of therapy are radical prostatectomy and radiotherapy (65). A review of prostate cancer therapies has shown that majority of patients receiving androgen-ablative or -deprivation therapy initially responded to treatment but became resistant over time and the cancer progressed (66).

In one study, the antiproliferative action of HC against a panel of prostate cancer cells comprising PC-3, C4-2, DU145 and 22Rv1 was shown to be dose- and time-dependent, with IC₅₀ values ranging from 30 to 320 µM (67). The HC treatment caused the PCa cells to accumulate in the G1 phase, signalling the onset of apoptosis (67), as evidenced by the upregulated levels of cleaved caspase-3 and cleaved PARP observed in PC-3 cells treated with HC for 18 h. HC was suggested to trigger cell death by generating high levels of ROS, which was further supported by a reduction in the mitochondrial transmembrane potential of PC-3 cells (67). Autophagic signalling was also postulated as an alternative pathway for the mechanism of action of HC in PCa cells since acidic vesicular organelles and the elevated expression of the autophagic markers light chain 3-IIb and beclin-1 were observed. Furthermore, the oral administration of 150 mg/kg reduced the tumour volume of PCa xenografts in mice by ~72% (67). In addition, the HC dosage was well tolerated with no signs of toxicity, and the IC₅₀ value of HC in RWPE normal prostate epithelial cells was 398 µM, which was much higher than that in the PCa cell lines (67).

As of 2020, CRC ranked third and second among the most common cancers in men and women, respectively (51,68). Chemotherapeutic drugs for CRC include 5-fluorouracil and oxaliplatin. However, over time multidrug resistance may develop in some patients (69). Therefore, the identification of alternative compounds with anticancer potential that can overcome the multidrug resistance pathway is crucial.

Similar to findings in other types of cancer (67), HC induced cell cycle arrest at the G0/G1 and G2/M phases in TP53-resistant HT-29 colon cancer cells, causing cells to accumulate in the G0/G1 phase (70). The apoptotic cell death observed in HT-29 cells following treatment with 30 µg/ml HC was higher than that of HT-29 cells treated with 50 µmol/l 5-fluorouracil for 12, 18, 24 and 30 h (70). The HT-29 cells treated with HC exhibited increased activation of JNK and p38 MAPK following 12 h of treatment while the 5-fluorouracil-treated cells did not (70).

Glioblastoma multiforme (GBM) is a primary malignant brain neoplasia that occurs in intracranial tissue or glial cells, which contribute to the supply of functional nutrients and oxygen to neurons (71). GBM patients typically have a poor prognosis, partly due to the infiltrative nature of glioma cells (72). Despite clinical and technological advances in the treatment of brain tumours over the last three decades, the survival of patients with GBM has not notably improved, and resistance to chemotherapy with agents such as temozolomide, is commonplace (73).

In an in vitro study, HC was shown to synergise with GTT, an isomer of vitamin E, to increase the death of glioma cells. This combined treatment activated caspase-3 by 7.1-79.0% and induced cell cycle arrest at the G2M and S-phases (74). In another study, the effect of HC and GTT in reducing the migration, invasion and colony formation of glioma cells was evidenced by a reduction in the downregulation of several genes in these pathways, namely cyclooxygenase (COX)-2, VEGFA, Jun and Wnt family member 5A (24). The ability of 1321N1, SW1783 and LN18 glioma cells to migrate was reduced by 16.1, 36.3 and 16.7%, respectively, when HC was combined with GTT (24,75). The ER-unfolded protein response (ER-UPR) pathway was postulated to contribute to the underlying mechanism for the HC and GTT combination. In addition, the expression of forkhead box protein M1, a crucial gene in pro-oncogenic signalling, was decreased in glioma by these combined treatments (24).

Liver cancer was ranked as the sixth most common cancer in Malaysia and worldwide and the third most common cause of cancer-associated death in Malaysia in 2020 (51). The risk factors for liver cancer include hepatitis B virus, hepatitis C virus, fatty liver disease, alcohol-associated cirrhosis, smoking, obesity, diabetes, iron overload and various dietary exposures (76). Most patients with liver cancer have a poor prognosis, with only 5–15% of patients at an early stage without cirrhosis being eligible for surgical excision (77). Patients with late-stage liver cancer are commonly treated with trans-arterial chemoembolisation (TACE) and the kinase inhibitor sorafenib as chemotherapy. As it the case with most chemotherapeutics, the long-term usage of TACE or sorafenib tends to cause side effects, toxicity and drug inefficacy (77).

A study revealed that the application of HC to HepG2 liver cancer cells pre-treated with BSO had cytotoxic effects, suggesting that the mechanism of action of HC was dependent on endogenous GSH. Although a low concentration of HC (12.5 µM) failed to reduce the viability of HepG2 cells when incubated for 24 h, 100 µM HC induced apoptosis over the same treatment period. These findings were confirmed by the observation of condensed chromatin by fluorescence microscopy and nuclear fragmentation using gel electrophoresis (78). It was concluded that these findings indicate that HC induces oxidative DNA damage and apoptosis in GSH-depleted HepG2 cells.

Oral cancer is one of the most common head-and-neck cancers, which includes cancers of the lip, tongue, gum, palate, mouth, floor of the mouth, gingiva and other parts of the oral cavity (79). In 2020, India had the highest number of oral cancer cases and Malaysia was ranked sixth among Southeast Asian countries for the prevalence of this type of cancer (51). Oral cancer may occur due to infection with human papillomavirus (HPV), poor hygiene, poor dental care and the consumption of unhealthy food (79). The most common treatment for oral cancer is surgical resection alone or combined with radiotherapy or adjuvant therapy. Despite the advancement of surgical techniques, the number of cases of oral cancer in certain Asian regions continues to rise, partly due to habits including alcohol consumption, smoking, tobacco chewing and areca nut chewing, as well as the limited availability of tertiary healthcare for most of the population (80–85).

A study indicated that at concentrations of <0.1 mM, HC exhibited anti-oxidative properties, while at higher concentrations, it inhibited oral KB carcinoma cell growth and cell cycle progression (31). Furthermore, at concentrations of 10 and 50 µM HC acted as a scavenger of H₂O₂, reducing H₂O₂-induced chemiluminescence by 53 and 75%, respectively (31). In addition, 0.02 µM HC effectively scavenged superoxide created by xanthine/xanthine oxidase (31). HC was a more potent scavenger of superoxide than of H₂O₂. The treatment of oral KB cells with ≥0.1 mM HC for 24 h induced cell cycle arrest at the late S and G2/M phases and reduced the GSH levels of the cells. Interestingly, at the low concentration of 0.01 mM, HC inhibited the production of ROS in oral KB cells. However, the intracellular accumulation of ROS occurred at a higher concentration of HC (0.1 mM) (31). In another study, 50 and 100 µM HC effectively inhibited the expression of matrix metallopeptidase (MMP-9) induced by areca nut extract in squamous cell carcinoma of the tongue epithelial cells. Although these concentrations of HC inhibited the expression of MMP-9, which plays a vital role in cancer invasion and metastasis, it could not halt the proliferation of the cells (86).

In a study of the effect of HC on KB epithelial cells (87), exposure to HC at concentrations of 0.1 and 0.3 mM for 6 h resulted in S-phase arrest. However, when treated with 0.1 mM HC for 24 h, some KB cells progressed to the G2/M and G0/G1 phases, and when exposed to 0.3 mM HC for 24 h, KB cells were either arrested in the S phase or became apoptotic (87). HC also induced mitochondrial depolarisation in the cells as demonstrated by impaired rhodamine uptake. At a concentration of 0.3 mM, HC caused GSH depletion and the generation of intracellular ROS in KB cells, while at concentrations of 0.1 and 0.2 mM, HC raised the GSH levels of the KB cells (87).

Skin cancer is a frequently occurring cancer for which sun exposure is the leading cause (88–90). Malignant melanoma (MM) and non-melanoma skin cancer (NMSC) are the two main types of skin cancer (88). Over the last 50 years, the incidence rates of both MM and NMSC have increased, with MM showing a 0.6% increase among adults (91).

In a study investigating the effect of HC on DNA biosynthesis in a female Swiss mice model of skin cancer, exposing the mice to 1 mg/day of HC demonstrated the ability to restore normal DNA biosynthesis in skin exposed to the carcinogen dimethylbenz[a]anthracene (92).

In summary, HC has shown anticancer effects in various cancer cell lines, including CML, glioma, breast, pancreatic, prostate, oral and colorectal cancer cells. In general, HC affects the JNK pathway, induces ROS, inhibits the cell cycle and lowers the viability of the cancer cells. However, data on the bioavailability of HC in cancer cells and animal models remains scarce. Bioavailability is the extent and rate at which an active compound enters the systemic circulation to reach the site of action (93). It remains unknown if HC goes directly to target sites or affects the organs, and the effects of HC in the body may be reduced as it migrates to the target site. Another aspect to consider is the inflammatory process that HC may induce in cancer cells. Unfortunately, little information is available on this in the literature, although the anti-inflammatory effects of HC have been studied in healthy cells. Table II summarises the anticancer activity of HC in various cancers based on in vitro and in vivo studies.