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Page 6 of 17 Wenner et al. J Cancer Metastasis Treat 2020;6:33 I http://dx.doi.org/10.20517/2394-4722.2020.73

the mitochondrial membrane potential and an increase in the ADP/ATP ratio, both indicative of decreased
cellular metabolic energy and autophagy. In addition, CAP increased concentrations of Atg4C, a protein
responsible for autophagosome development and upregulated autophagic genes: GABA Type A Receptor
Associated Protein Like 1 (GABARAPL1), LC3B, Sequestosome-1 (SQSTM1), immunity related GTPase M
(IRGM), Unc-51 like autophagy activating kinase 1 (ULK1), tumor necrosis factor (TNF) and phosphatase
and tensin homolog (PTEN). CAP-resistant cells underwent epithelial mesenchymal transition (EMT) and
autophagy through activation of the Hedgehog pathway when exposed to CAP. Interestingly, activation of
this pathway caused CAP-induced EMT cells to develop chemotherapeutic drug resistance to mitomycin C,
[39]
gemcitabine, and doxorubicin .

Studies have indicated that multiple signaling pathways mediate CAP-induced autophagy. Dihydrocapsaicin
(DHC), an analog of capsaicin, mediated autophagy in a catalase-dependent manner. Treatment
upregulated the expression of Atg5, a gene necessary for autophagosome formation, in addition to Atg4 and
Atg7 consequently increasing LC3-I conversion to LC3-II. Catalase induction by DHC decreased baseline
levels of ROS, consequently increasing production of LC3-II protein and caspase-3 activation. Upon
exposure to 3-amino-1, 2, 4-triazole (3AT), a catalase inhibitor, LC3-II levels from DHC treatment were
[40]
decreased. Conversely, overexpression of the catalase gene resulted in increased expression of LC3-II .
Lysosomes co-localized with LC3-II to form autolysosomes in treated LNCaP and PC-3 cells; however,
blockade of autophagy prevented their formation. High intracellular levels of ROS were observed in LNCaP
cells compared to PC-3 cells. Upon treatment with NAC, ROS production by CAP was decreased in both
cell lines. Confocal microscopy confirmed that cells treated with NAC did not accumulate autolysosomes
[41]
and reduced inhibition of the PI3K/Akt pathway, indicating the critical role of ROS in autophagy .
Another study confirmed that treated NPC-TW01 cells displayed reduced interaction between Beclin1
and Bcl-2, suggesting that autophagy activation may be regulated by the Beclin1/Bcl-2 complex and the
class III PI3K/Beclin1/Bcl-2 pathway. Inhibition of the PI3K/Akt/mTOR pathway suggested a correlation
between CAP and cellular proliferation . Table 2 summarizes the capsaicin-induced autophagic response
[42]
in different cancer models.

WITHAFERIN A
Withaferin A (WA), a steroidal lactone, is derived from a number of plants belonging to the family
Solanaceae, including Acnistus arborescens and Withania somnifera. It has been used in Indian medicine for
centuries and is currently being investigated in the Western world for its anticancer, antitumor, and anti-
inflammatory properties; however, its mechanism of action is unclear and is currently being investigated.

ROS generation was shown to be one of the main mechanisms by which WA prevents the growth of cancer
cells. For example, WA used alone or in combination with cisplatin has proven to induce cell death by
generating ROS and subsequent DNA damage in ovarian cancer and non-small cell lung cancer (NSCLC)
cells [43,44] . The combination of doxorubicinand WA in the treatment of ovarian cancer cell lines A2780,
A2780/CP70, and CaOV3 resulted in a time- and dose-dependent synergistic effect on the inhibition of
cell replication and induction of apoptosis. The combination treatment also showed enhancement of ROS
[45]
production, causing DNA damage . Colorectal cancer cells resistant to 5-FUthat were treated with the
combination of WA and 5-FU showed induction of ER stress, which eventually led to cell death. In these
cells, in addition to inducing G2M phase arrest, WA worked by upregulating stress sensors including BiP,
[46]
PERK, CHOP, ATF-4, and elF2a .
Hyperpolarization of the mitochondrial membrane is another mechanism of actionof WA. Targeting the
mitochondria would influence mitochondrial metabolic activity, which could eventually lead to paraptosis.
Cells treated with WA exhibited features (fusion of mitochondria and ER, expansion of vacuoles) that are
indicative of paraptosis. Another notable observation in the treated cells was a decrease in the expression
[47]
of Alix, anendogenous inhibitor of paraptosis . WA was shown to induce mitochondrial apoptosis