CASP3 Knockout Cell Line (HCT 116)

Although it has been established that cannabidiol (CBD), the major non-psychoactive constituent of cannabis, exerts antitumoral activities, the exact mechanism(s) via which tumor cells are killed by CBD are not well understood. This study provides new insights into the potential mechanisms of CBD-induced mutual antagonism of apoptosis and macroautophagy using wild type (HCT116 p53wt, LS174T p53wt), knockout (HCT116 p53) and mutant (SW480 p53mut) human colorectal cancer cells (CRC). CBD causes a more pronounced loss in the viability of p53wt cells than p53 and p53mut cells, and a 5-week treatment with CBD reduced the volume of HCT116 p53wt xenografts in mice, but had no effect on the volume of HCT116 p53 tumors. Mechanistically, we demonstrate that CBD only significantly elevates ROS production in cells harboring wild-type p53 (HCT116, LS174T) and that this is associated with an accumulation of PARP1. CBD-induced elevated ROS levels trigger G0/G1 cell cycle arrest, a reduction in CDK2, a p53-dependent caspase-8/9/3 activation and macroautophagy in p53wt cells. The ROS-induced macroautophagy which promotes the activation of keap1/Nrf2 pathway might be positively regulated by p53wt, since inhibition of p53 by pifithrin-α further attenuates autophagy after CBD treatment. Interestingly, an inhibition of heat shock protein 70 (Hsp70) expression significantly enhances caspase-3 mediated programmed cell death in p53wt cells, whereas autophagy-which is associated with a nuclear translocation of Nrf2-was blocked. Taken together, our results demonstrate an intricate interplay between apoptosis and macroautophagy in CBD-treated colorectal cancer cells, which is regulated by the complex interactions of p53wt and Hsp70.

The oncogenic BRAF(V600E) mutation activates the ERK1/2 pathway and is detected in 10% of human colorectal cancers (CRCs) where it is associated with poor prognosis. Inhibitors of BRAF have shown only modest efficacy in patients with CRC due to intrinsic drug resistance. We studied the CDK2/CDK9 inhibitor, fadraciclib, alone and in combination with the BRAF inhibitor encorafenib in isogenic human RKO CRC cells with two, one, or no BRAF alleles (RKO, A19, T29) and in BRAF wild-type HCT-116 cells, including Bax knockout HCT-116 cells. Treatment with fadraciclib was shown to suppress MCL-1 and phospho-MCL-1 (Ser64), induce a Bax-dependent apoptosis, and inhibit colony formation in a BRAF gene dose-dependent manner. Fadraciclib decreased phosphorylation of RNA polymerase II, indicating suppression of RNA transcription. The tumor growth inhibitory effect of fadraciclib plus encorafenib was synergistic. Fadraciclib decreased Rb phosphorylation, inhibited cell cycle progression, and promoted DNA damage as evidenced by cleavage of PARP, increased pH2AX (ser139), and activation of p53. In RKO versus A19 or T29 cells, drug treatment was associated with greater suppression of p-Rb and inhibition of apoptosis and the cell cycle. In a zebrafish xenograft model, fadraciclib plus encorafenib significantly reduced tumor size, concurrent with increased caspase-3 activation. In human CRCs, BRAF mutation was associated with overexpression of CDK2, and CDK9 overexpression was associated with worse patient survival. In conclusion, fadraciclib depletes MCL-1 to potentiate apoptosis and, combined with encorafenib, synergistically suppresses tumor cell growth in a BRAF gene dose-dependent manner. These data suggest a novel therapeutic strategy in CRCs with BRAF.

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

Alkaloids have garnered significant interest as potential anticancer agents. Vitamin D receptor (VDR) plays a role in preventing the progression of colorectal cancer (CRC) and may be a crucial mediator of the anticancer effects produced by certain alkaloids. The search for novel anticancer drugs that induce VDR expression and act through the VDR could improve the clinical outcomes of CRC patients. The anticancer and pro-apoptotic effects of coclaurine and reticuline were investigated using CRISPR/Cas9-edited VDR/knockout (KO) and wild-type (WT) CRC HCT116 cell lines. Western blotting, RT-qPCR, confocal microscopy, cell viability, scratch assays, and flow cytometry were employed to assess VDR expression and cellular localization, cell growth, wound-healing, cytotoxicity, apoptotic status, cell cycle progression, and VDR-mediated gene expression. Coclaurine and reticuline dose-dependently inhibited HCT116-WT cell viability, decreased wound-healing, and increased VDR nuclear localization and gene expression while downregulating the oncogenic genes and . Both alkaloids induced late apoptosis in HCT116-WT cells, increased the cleavage of PARP and caspase-3, and upregulated Bax and while decreasing . Both alkaloids caused HCT116-WT cell growth arrest in the S-phase, which is associated with cyclin A1 overexpression. Coclaurine and reticuline lost their anticancer effects in HCT116-VDR/KO cells. Docking studies revealed that both alkaloids occupied the VDR's active site. These findings demonstrate that coclaurine and reticuline exert anti-CRC and pro-apoptotic activities via the VDR, suggesting them as natural therapeutic candidates. The use of in vivo CRC models is needed to validate the anticancer activities of coclaurine and reticuline.