Novel quinazolinone MJ‑33 induces AKT/mTOR‑mediated autophagy‑associated apoptosis in 5FU‑resistant colorectal cancer cells

Abstract

Novel quinazolinone compounds have been studied in the field of drug discovery for a long time. Among their broad range of pharmacological effects, certain compounds effectively inhibit cancer cell proliferation. MJ‑33 is a quinazolinone derivative with proposed anticancer activities that was synthesized in our laboratory. The present study aimed to evaluate the anticancer activity of MJ‑33 in fluorouracil (5FU)‑resistant colorectal cancer cells (HT‑29/5FUR) and to investigate the underlying molecular mechanisms. The cell viability assay results indicated that HT‑29/5FUR cell viability was inhibited by MJ‑33 treatment in a concentration‑dependent manner compared with the control group. The cellular morphological alterations observed following MJ‑33 treatment indicated the occurrence of apoptosis and autophagy, as well as inhibition of cell proliferation in a time‑dependent manner compared with the control group. The acridine orange, LysoTracker Red and LC3‑green fluorescent protein staining results indicated that MJ‑33 treatment significantly induced autophagy compared with the control group. The DAPI/TUNEL dual staining results demonstrated increased nuclear fragmentation and condensation following MJ‑33 treatment compared with the control group. The Annexin V apoptosis assay and image cytometry analysis results demonstrated a significant increase in apoptotic cells following MJ‑33 treatment compared with the control group. The western blotting results demonstrated markedly decreased Bcl‑2, phosphorylated (p)‑BAD, pro‑caspase‑9 and pro‑caspase‑3 expression levels, and notably increased cytochrome c and apoptotic peptidase activating factor 1 expression levels following MJ‑33 treatment compared with the control group. Moreover, the expression levels of autophagy‑related proteins, including autophagy related (ATG)‑5, ATG‑7, ATG‑12, ATG‑16, p62 and LC3‑II, were increased following MJ‑33 treatment compared with the control group. Furthermore, MJ‑33‑treated HT‑29/5FUR cells displayed decreased expression levels of p‑AKT and p‑mTOR compared with control cells. The results suggested that MJ‑33‑induced apoptosis was mediated by AKT signaling, and subsequently modulated via the mitochondria‑dependent signaling pathway. Therefore, the results suggested that suppression of AKT/mTOR activity triggered autophagy in the HT‑29/5FUR cell line. In summary, the results indicated that MJ‑33 inhibited HT‑29/5FUR cell viability, and induced apoptosis and autophagy via the AKT/mTOR signaling pathway. The present study may provide novel insight into the anticancer effects and mechanisms underlying MJ‑33 in 5FU‑resistant colorectal cancer cells.

Keywords: MJ‑33; colorectal cancer; fluorouracil‑resistant HT‑29 cells; autophagy; apoptosis.

Figure 1.

Effects of MJ-33 on HT-29/5FUR and CCD 841 CoN cell viability. Cells were seeded (1×10⁴ cells/well) into 96-well plates and treated with 0, 25, 50, 75 and 100 µM MJ-33. HT-29/5FUR cell viability following treatment for (A) 24 or (B) 48 h. CCD 841 CoN cell viability following treatment for (C) 24 or (D) 48 h. Data are presented as the mean ± SD from three independent experiments. Data were analyzed using one-way ANOVA followed by Dunnett's post hoc test. ***P<0.001 vs. control. 5FUR, fluorouracil-resistant; Ox, oxaliplatin.

Figure 2.

Effects of MJ-33 on HT-29/5FUR cell morphology. (A) Morphological alterations and cell death in HT-29/5FUR cells following treatment with MJ-33 (magnification, ×200). (B) Apoptotic and autophagic cells were observed. 5FUR, fluorouracil-resistant (magnification, ×200).

Figure 3.

MJ-33 treatment induces HT-29/5FUR cell apoptosis. (A) DNA condensation and apoptotic DNA fragmentation were observed by performing DAPI and TUNEL staining in MJ-33-treated HT-29/5FUR cells (magnification, ×200). (B) Quantification of TUNEL-positive cells. (C) Cell apoptosis was assessed by performing Annexin V/PI dual staining via image cytometry in MJ-33-treated HT-29/5FUR cells. (D) Quantification of Annexin V⁺/PI⁻ cells. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. ***P<0.001. 5FUR, fluorouracil-resistant.

Figure 4.

MJ-33-induced HT-29/5FUR cell apoptosis via initiation of the caspase cascade. Cell viability following treatment with MJ-33 and/or (A) pan-caspase inhibitor (z-VAD-FMK), (B) caspase-9 inhibitor (z-LEHD-FMK) or (C) caspase-3 inhibitor (z-DEVD-FMK). Data are presented as the mean ± SD from three independent experiments. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. ***P<0.001; ^###P<0.001. 5FUR, fluorouracil-resistant.

Figure 5.

MJ-33 activated the intrinsic apoptosis signaling pathway. Western blotting was performed to measure the expression levels of (A) proapoptotic and (B) intrinsic apoptosis signaling pathway-related proteins. The density of the bands compared with the control sample (set to 1.0) are presented above each band. p, phosphorylated; Apaf-1, apoptotic peptidase activating factor 1.

Figure 6.

MJ-33 induced HT-29/5FUR cell autophagy. (A) AO, LysoTracker Red and LC3-GFP staining of MJ-33-treated HT-29/5FUR cells was observed under a fluorescence microscope (magnification, ×200). Relative fluorescence intensity of (B) AO, (C) LysoTracker Red uptake and (D) LC3-GFP. Data are presented as the mean ± SD from three independent experiments. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. ***P<0.001. 5FUR, fluorouracil-resistant; AO, acridine orange; GFP, green fluorescent protein.

Figure 7.

Effects of MJ-33 treatment on the expression levels of AKT/mTOR axis- and autophagy-related proteins in HT-29/5FUR cells. Western blotting was performed to measure the expression levels of (A) AKT/mTOR axis- and (B) autophagy-related proteins in HT-29/5FUR cells following MJ-33 treatment. The density of the bands compared with the control sample (set to 1.0) are presented above each band. 5FUR, fluorouracil-resistant; p, phosphorylated; ATG, autophagy related.

Figure 8.

Involvement of autophagy in MJ-33-treated HT-29/5FUR cells. Cell viability following treatment with MJ-33 and/or (A) CQ, (B) 3-MA and (C) Baf.A. Data are presented as the mean ± SD from three independent experiments. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. ***P<0.001; ^###P<0.001. 5FUR, fluorouracil-resistant; CQ, chloroquine; 3-MA, 3-methyladenine; Baf.A, bafilomycin A1.

Figure 9.

MJ-33-induced apoptosis is regulated via an autophagy mechanism and AKT activity. (A) Effect of 3-MA on caspase-3 and caspase-7 activities in MJ-33-treated HT-29/5FUR cells. (B) Effect of SC-79 on HT-29/5FUR cell viability following treatment with MJ-33. Data are presented as the mean ± SD from three independent experiments. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. ***P<0.001; ^###P<0.001. 3-MA, 3-methyladenine; 5FUR, fluorouracil-resistant.

Figure 10.

Schematic diagram of the proposed mechanism underlying MJ-33-induced apoptosis and autophagy in fluorouracil-resistant HT-29 cells. p, phosphorylated; ATG, autophagy related; 3-MA, 3-methyladenine; LC3, microtubule associated protein 1 light chain 3α; CQ, chloroquine; Baf.A, bafilomycin A1; Apaf-1, apoptotic peptidase activating factor 1.