*6.1. Anti-Cancer Activity of PTS*

Several experimental studies have demonstrated the inhibitory effects of PTS against various cancer cells, including stomach, skin, lung, liver, breast, colon, pancreas, oral, lymph, cervical, endometrial, hematological melanoma, prostate, leukemia, and myeloma tumor cells [29,30] (Table 2). PTS has been identified to be useful in preventing and treating cancer by regulating pro-apoptotic or non-apoptotic anti-cancer activities [33]. The development and progression of cancer involve various factors: a group of drugmetabolizing enzymes, cytochrome P450. These enzymes mediate the metabolic activation of several pro-carcinogens and play a crucial role in the inactivation and activation of anti-cancer drugs. CYP1A1 and CYP1B1 are members of the CYP1 superfamily and have important roles in cancer progression. PTS acts as an efficient inhibitor of CYP1A1, CYP1A2, and CYP1B2 in a competitive manner. The anti-proliferative mechanisms of PTS are seen in different concentrations for different cell types [3].


**Table 2.** Effect of Pterostilbene in Experimental Models of Various Disease Conditions.



Rimando et al. studied the cancer chemopreventive activity of PTS using a mouse mammary gland culture model and showed that PTS (ED50 = 4.8 μM) markedly reduced DMBA-induced mammary alveolar precancerous lesions through its peroxy-radical scavenging antioxidant activity [62]. In mutant p53-breast cancer cell lines MDA-MB-231 and T-47D, PTS facilitated the reduction in oncogenic β-catenin, mTOR, and mutant p53, as well as increased the expression of the pro-apoptotic Bax protein [87]. Moreover, in MDA-MB-231 xenograft mouse models, PTS suppressed the epithelial-to-mesenchymal transition (EMT) through the upregulation of miR-205 and the consequent reduction in pro-EMT src signaling [63]. Notably, PTS exhibited additive anti-cancer effects in combination with other natural compounds. A combination of α-tocopherol succinate (42 and 99 IU/kg) and PTS (40 μg/kg) attenuated the invasive capability of MDA-MB-231 cells [68]. The treatment of ER-positive breast cancer with a combination of tamoxifen (5 μM) and PTS (10 and 20 μM) also exhibited additive effects. In the studied cell lines (MCF7 and ZR-751), the suppression of cancer cell proliferation along with an elevation in apoptotic activity was observed when this combination was employed [45].

In the human colorectal adenocarcinoma cell line HT-29, PTS (≥10 μM) inhibited cell proliferation while inducing G1 cell arrest. Moreover, PTS treatment stimulated apoptosis through the attenuation of the STAT3 and AKT kinase signaling pathways [88]. In a rodent model of azoxymethane (AOM)-induced colon cancer, the intake of PTS (40 ppm) through the diet for 45 weeks led to a reduction in tumorigenesis and diminished the levels of proliferating cell nuclear antigen (PCNA), cyclin D1, and β-catenin [89]. PTS was also observed to mediate the anti-cancer effect through the stimulation of Nrf2 signaling and its target genes (HO-1 and GR), which counter the effect of NF-κB-mediated pro-inflammatory signaling. Studies have shown that the overexpression of iNOS and COX-2 is markedly correlated with the progression of colon cancer. Moreover, in an in vitro study using the HT-29 colon cancer model, PTS inhibited the transcriptional expression of augmented iNOS levels and moderated the inhibition of COX-2 in a concentration-dependent manner [40,89].

PTS in combination with quercetin at 20 mg/kg/day inhibited the metastatic activity in B16-F10 melanoma by reducing the adhesion of B16-F10 cells to the endothelium and also downregulated the levels of Bcl-2 in cancerous cells [90]. PTS (10 to 50 μM) suppressed the cancer cell proliferation and initiated apoptotic signaling through the induction of lysosomal membrane permeabilization in A375 melanoma cells [91]. Moreover, through the attenuation of iNOS and COX-2 expression, PTS prevented DMBA- and TPAinduced skin tumor formation [92]. Similarly, in rodent models of UVB-induced skin cancer, the anti-cancer activity of PTS was observed to include the prominent induction of Nrf2-mediated antioxidant signaling, resulting in glutathione level maintenance and the improved activities of catalase, SOD, and GPX [41]. Intravenously administered PTS suppresses human melanoma and pancreatic cancer growth in small animals. Evidence indicates an indirect mechanism of cancer growth inhibition, where PTS inhibits pituitary adrenocorticotropic hormone production, mediates the downregulation of glucocorticoid receptors, and stimulates the Nrf2-dependent cancer antioxidant defense system and the stress-related neuroendocrine signaling mechanism [30,69].

Multiple myeloma (MM) models, xenograft mouse models for hematological cancers, and several diffuse large B-cell lymphoma (DLBCL) models have been utilized to study the anti-cancer effect of PTS [64,65]. In the DLBCL cell line, the viability of the cancer cells was largely dependent on the concentration of PTS and was associated with reduced mitochondrial membrane potential, elevated free-radical generation, and caspase-mediated apoptosis when PTS was intravenously administered [64]. In MM cell lines, a similar concentration-dependent suppression of the proliferation of cancer cells was observed through increased caspase activation, further highlighting the anti-cancer properties of PTS [65]. Additionally, PTS treatment was reported to show benefits against Cholangiocarcinoma (CCA), also known as biliary tract cancer, as evidenced by its cytotoxic effects, mediated through autophagy and the inhibition of CCA tumor growth, in two different CCA cell lines [70].

In endometrial cancer cells, the combination of PTS and megestrol acetate produced a synergistic effect through the inhibition of cell-cycle regulators, including cyclin D1, cyclin B1, and CDK4 [93]. An open-label randomized Phase II clinical trial is underway to study the effect of PTS with megestrol acetate in endometrial cancer patients or patients with endometrial complex atypical hyperplasia who are scheduled for hysterectomy (ClinicalTrials.gov Identifier: NCT03671811) (Table 3). Furthermore, PTS suppresses cell-cycle progression and apoptosis in ovarian cancer cells (OVCAR-8 and Caov-3 cells) through the inhibition of the STAT3 pathway. PTS decreased the expression of cell-cycle and antiapoptotic proteins involved in the STAT3 pathway, including Mcl-1, Bcl-2, and cyclin D1 [94].


**Table 3.** Clinical Trials Involving Pterostilbene.


There are scientific reports on the therapeutic potential of PTS in attenuating hepatocellular carcinoma (HCC), which is the second-most prominent cause of cancer-related mortality. In a recent study, PTS treatment was reported to inhibit tumor growth and cell proliferation in a dose-dependent manner in an animal model of HCC [96]. A combination of diethylnitrosamine and carbon tetrachloride was used to induce HCC in mouse livers. PTS treatment was demonstrated to upregulate caspase-3 activity and thereby induce apoptosis in HCC tumor tissue. Interestingly, PTS was identified to reduce HCC proliferation through a reduction in SOD2 and the induction of ROS-mediated mitochondrial apoptotic pathways [96]. Further, it was observed that PTS conferred protection against HCC proliferation and inhibited Hepatitis B virus proliferation in several HCC cell lines. Of note, PTS exhibited antiviral and anti-cancer activity in HCC cells that were resistant to Sorafenib (anti-cancer drug) and Lamivudine, an antiretroviral drug [97]. Importantly, the researchers identified that PTS exhibited anti-cancer and anti-retroviral effects through the potent inhibition of ribonucleotide reductase (RR), which plays a critical role in cellular DNA synthesis. Additionally, PTS treatment was demonstrated to markedly inhibit the growth of an HCC xenograft in nude mice with minimal toxicity [97]. PTS was reported to suppress the invasion and growth of HCC by down-regulating the expression of Metastasis-Associated Protein 1 (MTA1) and histone deacetylase 1 (HDAC1) while upregulating the acetylation of the tumor suppressor protein PTEN [98]. The epigenetic-level regulation by PTS could open new avenues in understanding its anti-cancer activity.
