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Wenner et al. J Cancer Metastasis Treat 2020;6:33 I http://dx.doi.org/10.20517/2394-4722.2020.73 Page 5 of 17

CCA cellsexamined, QBC939, SK-ChA-1, and MZ-ChA-1, exhibited MDR to the chemotherapeutic drugs
cisplatin, vincristine, and 5-fluorouracil (5-FU) as well as CAP. In vitro, combined treatment of 5-FU with
low concentrations of CAP displayed synergistic effects in 5-FU-induced anti-proliferation, increasing
sensitivity to apoptosis. The autophagy inhibitor 3-methyladenine (3-MA) enhanced the effectiveness
of 5-FU inQBC939 cells, while the autophagy activator retinoic acid receptor alpha (RARA) promoted
resistance to 5-FU. Studies have shown that the growth and survival effects of autophagy typically promote
chemotherapeutic drug resistance. 5-FU treatment increased the expression of Beclin 1, LC3-II, and Atg5
genes as evidenced by acridine orange staining, PCR, and Western blot analysis. CAP/5-FU co-treatment
inhibited 5-FU-mediated autophagy through phosphorylation of the Akt/mTOR pathway. Hence, CAP
[33]
hindered chemotherapeutic drug resistance, proving its potential as an adjunct to conventional therapies .
In response to environmental stresses such as DNA damage, CAP increases cellular viability through
autophagy in certain cell lines. One study explored the impact of CAP on glioblastoma and breast cancer
cell lines with mutated p53 oncosuppressor genes. Mutated p53 cells are known to express resistance to
common therapies and promote tumor growth. The study noted that CAP increased levels of LC3-II,
reduced p62 protein, and reduced mutant p53 by autophagy-mediated protein degradation. In addition,
treatment caused p53 to transactivate damage-regulate autophagy modulator, a gene that induces autophagy
through lysosomal protein expression, among other apoptotic genes. Western blotting confirmed that
poly ADP-ribose polymerase (PARP) cleavage occurred after CAP treatment in both U373 and DKBR3
cells. The loss of mutant p53 inevitably altered the ratio of folded to misfolded p53 proteins, consequently
[34]
restoring wild-type p53 protein activity in target cells . CAP-induced autophagy has also been shown to
repair DNA in MCF-7 breast cancer cells. Autophagy was initiated by the AMPKα-mTOR pathway and
the concentration of p53 protein increased in both the nucleus and cytosol. Moreover, cells experienced
an alteration of mitochondrial membrane potential and blockingS-phase. In response to cancerous gene
damage, CAP initiated DNA repair by activating δ-H2AX, ATM, DNA-PKcs, and PARP-1. Inactivation
of the Atg5 gene in M059K cells resulted in phosphorylation of ATM and DNA-PKcs and increased
expression of p53 and LC3-II. ATM and p53 inhibition resulted in PARP cleavage and prevented DNA-PKcs
phosphorylation without changing LC3-II levels. Therefore, autophagy inducedby genetic damage
[35]
improved cellular viability by repairing DNA through ATM regulation of DNA-PKcs and PARP-1 .
Inhibition of CAP-induced autophagy can enhance cellular sensitivity to apoptosis in a variety of
human cancers. CAP decreased cellular viability in U251 glioma cells by increasing the expression of
Beclin1, p62, and PUMA. Treatment with 3-MA, a known inhibitor of PI3K that blocks the formation of
autophagosomes, reduced procaspase-3 and Beclin1 while increasing the expression of p53 and PUMA.
Inhibition of p53 increased the expression of autophagic proteins. Therefore, inhibition of CAP-induced
autophagy in glioma cells contributed to apoptosis . As discussed above, CAP synergistically restricted
[36]
proliferation, increasing CCA cell sensitivity to apoptosis and upregulated apoptotic gene expression upon
autophagy inhibition in OS cells [32,33] . Multiple studies have demonstrated that CAP promotes apoptosis in a
time-dependent manner upon inhibition of autophagy in hepatocellular carcinoma, melanoma and bladder
cancercells [37-39] . CAP was shown to target the ROS/signal transducer and activator of transcription 3 (Stat3)
pathway in hepatocellular carcinoma cells. Western blotting indicated increased expression of autophagy
markers LC3-II and Beclin1, GFP-LC3 autophagosome formation, ROS generation, and upregulated
phosphorylation of Stat3, a known regulator of autophagy. In addition, NAC reversed the effects of CAP
on ROS/Stat3-dependent autophagy, consequently inducing apoptosis . CAP stimulated LC3-II levels,
[36]
formation of acidic vesicular organelles, and expression of ubiquitin-binding protein p62, which transports
other proteins to the phagophore complex for selective autophagy, in 5,637 and T24 bladder cancer cells.
To test if CAP could induce autophagy at varying levels of p62, cells were co-treated with CAP and BAF,
a lysosomal inhibitor. Cells were found to generate p62, proving that CAP can activate autophagic flux. It
was noted that CAP treatment generated ROS production resulting in time-dependent depolarization of