Although the life expectancy of colorectal cancer (CRC) patients substantially increased by development of combinational chemotherapy, the main challenges in treating CRC patients are the harmful effects of these agents on healthy tissues and the development of drug resistance by tumor cells (27). In the past few years, all these limitations have been overcome by advancements in DNA microarrays, proteomics technology, and targeted therapies (28). PI3K/AKT signaling is one of the most important cellular pathways, and the consecutive activation of this pathway in the CRC has been found to be related to the progression of the tumor, the survival of the cells, and drug resistance (29). Currently, significant progresses in the design of small molecule inhibitors for different classes of PI3K has been made and many studies and clinical trials are evaluating novel drugs that interfere with components of the PI3K/AKT axis (30). To date, several PI3K/AKT signaling inhibitors have been made which can be categorized into isoform specific, dual or pan inhibitors (31). Among different members of the selective small molecule inhibitors of PI3K signaling, BKM120 (buparlisib), a pan-class I PI3K inhibitor, gained a lot of attention due to its great antitumoral effects. Currently, clinical efficacy of BKM120 is evaluating for the treatment of different adult cancers including prostate, breast, advanced non-small cell lung cancers, and advanced solid tumors (32–35). In the current study, we assessed the effects of BKM120 in colorectal cancer-derived SW480 cells and we tried to uncover the exact mechanisms by which as PI3K inhibition exert its anti-cancer effects.
Based on the results of our experiments, BKM120 could markedly diminished the metabolic activity of SW480 cells in a concentration and time-dependent manner and estimated IC50 value was about 4.1 µM. In harmony with our result, Mueller et al. declared that BKM120 substantially reduced the survival of both colorectal cancer cells (HT29, HCT-116 and DLD-1) and gastric cancer cell (MKN-45, NCIn87 and AGS). With an IC50 of 1 µM, the colon cancer cell lines were more susceptible to the effects of BKM120 than the gastric cancer cell lines, which needed at least 2–5 µM (36). Considering the role of PI3K/AKT in regulation of cell cycle, we assessed the distribution of BKM120-treated cells in cell cycle phases. The result of cell cycle analysis showed that BKM120 increased the percentage of cells in G2/M phase, suggesting that BKM120-mediated anti-cancer effect is mediated through a G2/M arrest. Consistently, Koul et al. showed that blockade of PI3K/AKT activity by BKM- 120 induced G2–M cell cycle arrest in glioma cells (37). Roy et al. reported that PI3K/AKT Inhibition causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer. Notably, the researchers found that FOXO plays an important role in regulating cell cycle and apoptotic genes like p27 and p21, which are cyclin-dependent kinase inhibitors (38). In line with these evidences, our result showed that BKM120 mediated G2/M cell cycle arrest in SW480 cells was coupled with a robust increase in the expression of FOXO3a, p21 and p27 genes, suggesting that these key regulators of cell cycle could hinder the transition of cells from G2/M phase. Moreover, evaluation the expression of SIRT1 which governs cell-cycle progression by modulating acetylation and phosphorylation of checkpoint kinase 2 (CHK2), was dramatically decreased in treated SW480 cells (39).
Results of annexin IV/PI staining and flowcytometric assay showed that anticancer effects of BKM120 was mediated though apoptosis which this result was confirmed by a significant alteration in the expression of Bcl-2 family members. In agreement, in a study conducted by Civallero et al., it has been reported that BKM120 decreased the survival of lymphoma cell lines through regulation of Bcl-2 family members in favor of apoptosis induction (40). Mounting body of evidence has reported that autophagy play a crucial regulatory role in drug-induced apoptosis and can promote either cancer cell survival or death (41). However, the regulatory effect of autophagy on BKM120-mediated apoptosis in SW480 cells is still not elucidated. Interestingly, elevation the mRNA level of autophagy-associated genes such as ATG7, ATG10, and Beclin-1, which are essential for formation of autophagosomal structures (42), showed that autophagy was activated in BKM120-treated SW480 cells. Then, we speculated that the pharmacological inhibition of autophagy, obtained with chloroquine (CQ) administration, could affect on the anti-cancer effects of BKM120. Notably, autophagy inhibition could reduce the survival of SW480 cells, either as a single agent or in combination with BKM120, indicating that the activation of autophagy system could attenuate anticancer effects of PI3K inhibitor on SW480 cells. Based on the fact that PI3K/AKT signaling can promote cancer invasion and metastasis through its downstream activated proteins (26), we were interested to determine the influences of PI3K inhibition on invasion capacity of SW480 cells. According to the result of the scratch assay, BKM120-treated cells did not fill the scratch area which declare that PI3K inhibition led to the suppression of proliferation and invasion in SW480 cells. Since matrix metalloproteinase (MMPs) including MMP2 and MMP9, vascular endothelial growth factor (VEGF), and collagen are the main weapons of cancer cells to degrade the extracellular matrix and other barriers, angiogenesis and migration, we evaluated the mRNA level of these genes (43). Based on our result, expression of MMP2, MMP9, VEGF, and COL1A1 dramatically decreased in BKM120-treated cells in comparison to untreated cells, which further highlight the results of scratch assay (Fig. 7).
Since it has been well established that PI3K/AKT pathway can play a prominent role in chemoresistance against Cisplatin, as one of the most potent chemotherapeutic agents for the treatment of CRC (44, 45), we aimed to determine the effects of PI3K inhibition on the cytotoxicity of Cisplatin in SW480 cells. Combinatorial experiments demonstrated that BKM120 sensitized SW480 cell to lower concentrations of Cisplatin, which highlights the fact that chemotherapeutic potentials of Cisplatin could be weakened, at least partially, through PI3K/AKT axis activity. In conclusion, our preclinical study showed promising anti-cancer potentials of PI3K inhibition against colorectal cancer-derived SW480 cells and disclosed the exact mechanism underlying the efficacy of BKM120. Since BKM120 is an ongoing drug in the therapeutic approaches of human cancers, the results of this study shed new light on the potentials of this small molecule inhibitor for treatment of CRC patients. However, further preclinical and clinical studies are required to confirm this claim.