Kushal Kandhari

The anticancer potential and associated mechanisms of flavonoid fisetin are yet to be fully investigated on human head and neck squamous cell carcinoma (HNSCC). In the present study, fisetin (25‐75 µM for 24–48 h) dose‐dependently inhibited growth and induced death in HNSCC Cal33 and UM‐SCC‐22B cells, without showing any death in normal cells. Fisetin (25–50 µM) induced G2/M phase arrest via decrease in Cdc25C, CDK1, cyclin B1 expression, and an increase in p53^(S15). A concentration‐dependent increase in fisetin‐induced DNA damage and apoptosis in HNSCC cells was authenticated by comet assay, gamma‐H2A.X^(S139) phosphorylation, and marked cleavage of PARP protein. Interestingly, fisetin‐induced cell death occurred independently of p53 and reactive oxygen species production. The activation of JNK and inhibition of PI3K/Akt, ERK1/2, EGFR, and STAT‐3 signaling were identified. Further, fisetin‐induced apoptosis was mediated, in part, via p21^Cip1 and p27^Kip1 cleavage by caspase, which was reversed by z‐VAD‐FMK, a pan‐caspase inhibitor. Subsequently, fisetin was also found to induce autophagy; nevertheless, autophagy attenuation exaggerated apoptosis. Oral fisetin (50 mg/kg body weight) treatment to establish Cal33 xenograft in mice for 19 days showed 73% inhibition in tumor volume (p < 0.01) along with a decrease in Ki67‐positive cells and an increase in cleaved caspase‐3 level in tumors. Consistent with the effect of 50 µM fisetin in vitro, the protein levels of p21^Cip1 and P27^Kip1 were also decreased by fisetin in tumors. Together, these findings showed strong anticancer efficacy of fisetin against HNSCC with downregulation of EGFR‐Akt/ERK1/2‐STAT‐3 pathway and activation of JNK/c‐Jun, caspases and caspase‐mediated cleavage of p21^Cip1 and p27^Kip1.

Silybin is a natural compound extensively studied for its hepatoprotective, neuroprotective and anticancer properties. Envisioning the enhancement of silybin potential by suitable modifications in its chemical structure, here, a series of new 7-O-alkyl silybins derivatives were synthesized by the Mitsunobu reaction starting from the silybins and tyrosol-based phenols, such as tyrosol (TYR, 3), 3-methoxytyrosol (MTYR, 4), and 3-hydroxytyrosol (HTYR, 5). This research sought to explore the antioxidant and anticancer properties of eighteen new derivatives and their mechanisms. In particular, the antioxidant properties of new derivatives outlined by the DPPH assay showed a very pronounced activity depending on the tyrosyl moiety (HTYR > MTYR >> TYR). A significant contribution of the HTYR moiety was observed for silybins and 2,3-dehydro-silybin-based derivatives. According to the very potent antioxidant activity, 2,3-dehydro-silybin derivatives 15ab, 15a, and 15b exerted the most potent anticancer activity in human prostate cancer PC-3 cells. Furthermore, flow cytometric analysis for cell cycle and apoptosis revealed that 15ab, 15a, and 15b induce strong G1 phase arrest and increase late apoptotic population in PC-3 cells. Additionally, Western blotting for apoptotic marker cleaved caspase-3 confirmed apoptosis induction by these silybin derivatives in PC-3 cells. These findings hold significant importance in the investigation of anticancer properties of silybin derivatives and strongly encourage swift investigation in pre-clinical models and clinical trials.

Silybin is a natural compound extensively studied for its hepatoprotective, neuroprotective and anti-cancer properties. Envisioning the enhancement of silybin potential by suitable modifications in its chemical structure, here, a series of new 7-O alkyl silybins derivatives were synthesized by Mitsunobu reaction starting from the silybins and tyrosol-based phenols, such as tyrosol (TYR, 3), 3-methoxytyrosol (MTYR, 4) and 3-hydroxytyrosol (HTYR, 5). This research sought to explore the antioxidant and anticancer properties of new eighteen derivatives and their mechanisms. In particular, the antioxidant properties of new derivatives outlined by DPPH assay, showed a very pronounced activity depending on the tyrosyl moiety (HTYR>MTYR>>TYR). A significant contribution of the HTYR moiety was observed for silybins and 2,3-dehydro-silybin based derivatives. According to the very potent antioxidant activity, 2,3-dehydro-silybin derivatives 15ab, 15a and 15b exerted the most potent anticancer activity in human prostate cancer PC-3 cells. Furthermore, flow cytometric analysis for cell cycle and apoptosis revealed that 15ab, 15a and 15b induce strong G1 phase arrest and increase late apoptotic population in PC-3 cells. Additionally, western blotting for apoptotic marker cleaved caspase-3 confirmed apoptosis induction by these silybin derivatives in PC-3 cells. These findings hold significant importance in the investigation of anticancer properties of silybin derivatives and strongly encourage swift investigation in pre-clinical models and clinical trials.

The consumption of the non-steroidal anti-inflammatory drug (NSAID) aspirin is associated with a significant reduction in the risk of developing TMPRSS2-ERG (fusion)-positive prostate cancer (PCa) compared to fusion-negative PCa in population-based case–control studies; however, no extensive preclinical studies have been conducted to investigate and confirm these protective benefits. Thus, the focus of this study was to determine the potential usefulness of aspirin and another NSAID, naproxen, in PCa prevention, employing preclinical models of both TMPRSS2-ERG (fusion)-driven (with conditional deletion of Pten) and non-TMPRSS2-ERG-driven (Hi-Myc^+/− mice) PCa. Male mice (n = 25 mice/group) were fed aspirin- (700 and 1400 ppm) and naproxen- (200 and 400 ppm) supplemented diets from (a) 6 weeks until 32 weeks of Hi-Myc^+/− mice age; and (b) 1 week until 20 weeks post-Cre induction in the fusion model. In all NSAID-fed groups, compared to no-drug controls, there was a significant decrease in higher-grade adenocarcinoma incidence in the TMPRSS2-ERG (fusion)-driven PCa model. Notably, there were no moderately differentiated (MD) adenocarcinomas in the dorsolateral prostate of naproxen groups, and its incidence also decreased by ~79–91% in the aspirin cohorts. In contrast, NSAIDs showed little protective effect against prostate tumorigenesis in Hi-Myc^+/− mice, suggesting that NSAIDs exert a specific protective effect against TMPRSS2-ERG (fusion)-driven PCa.

Herein, we assessed the stage-specific efficacy of inositol hexaphosphate (IP6, phytic acid), a bioactive food component, on prostate cancer (PCa) growth and progression in a transgenic mouse model of prostate cancer (TRAMP). Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water for ~8–15 weeks. Pathological assessments at study endpoint indicated that tumor grade is arrested at earlier stages by IP6 treatment; IP6 also prevented progression to more advanced forms of the disease (~55–70% decrease in moderately and poorly differentiated adenocarcinoma incidence was observed in advanced stage TRAMP cohorts). Next, we determined whether the protective effects of IP6 are mediated via its effect on the expansion of the cancer stem cells (CSCs) pool; results indicated that the anti-PCa effects of IP6 are associated with its potential to eradicate the PCa CSC pool in TRAMP prostate tumors. Furthermore, in vitro assays corroborated the above findings as IP6 decreased the % of floating PC-3 prostaspheres (self-renewal of CSCs) by ~90%. Together, these findings suggest the multifaceted chemopreventive-translational potential of IP6 intervention in suppressing the growth and progression of PCa and controlling this malignancy at an early stage.

In the present study, we performed a comparative stage‐specific pathological and molecular marker evaluation of TMPRSS2‐ERG fusion and PTEN loss‐driven (TMPRSS2‐ERG. Pten^flox/flox) versus non‐fusion‐driven prostate tumorigenesis (Hi‐Myc) in mice. Anterior, ventral, and dorsolateral prostates were collected from mice at different ages (or time points post‐Cre induction). Results indicated that growth and progression of prostatic intraepithelial lesions to adenocarcinoma stages occurred in both mice models albeit at different rates. In the TMPRSS2‐ERG. Pten^flox/flox mice, the initiation of tumorigenesis was slow, but subsequent progression through different stages became increasingly faster. Adenocarcinoma stage was reached early on; however, no high‐grade undifferentiated tumors were observed. Conversely, in the Hi‐Myc^+/− mice, tumorigenesis initiation was rapid; however, progression through different stages was relatively slower and it took a while to reach the more aggressive phenotype stage. Nevertheless, at the advanced stages in the Hi‐Myc^+/− mice, high‐grade undifferentiated tumors were observed compared to the later stage tumors observed in the fusion‐driven TMPRSS2‐ERG. Pten^flox/flox mice. These results were corroborated by the stage specific‐pattern in the molecular expression of proliferation markers (PCNA and c‐Myc); androgen receptor (AR); fusion‐resultant overexpression of ERG; Prostein (SLC45‐A3); and angiogenesis marker (CD‐31). Importantly, there was a significant increase in immune cell infiltrations, which increased with the stage of tumorigenesis, in the TMPRSS2‐ERG fusion‐positive tumors relative to fusion negative tumors. Together, these findings are both novel and highly significant in establishing a working preclinical model for evaluating the efficacy of interventions during different stages of tumorigenesis in TMPRSS2‐ERG fusion‐driven PCa.

Kushal Kandhari, Sandeep Paudel, Komal Raina, Chapla Agarwal, Rama Kant, Michael F. Wempe, Cindy O’Bryant, Rajesh Agarwal. J Cancer Prev 2021;26:266-76. https://doi.org/10.15430/JCP.2021.26.4.266