DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) inhibits chemotherapeutic drug-induced autophagy

Abstract

Our previous work found that DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl]quinoline) was a novel autophagy inhibitor. Here, we aimed to investigate the effects of DMH1 on chemotherapeutic drug-induced autophagy as well as the efficacy of chemotherapeutic drugs in different cancer cells. We found that DMH1 inhibited tamoxifen- and cispcis-diaminedichloroplatinum (II) (CDDP)-induced autophagy responses in MCF-7 and HeLa cells, and potentiated the anti-tumor activity of tamoxifen and CDDP for both cells. DMH1 inhibited 5-fluorouracil (5-FU)-induced autophagy responses in MCF-7 and HeLa cells, but did not affect the anti-tumor activity of 5-FU for these two cell lines. DMH1 itself did not induce cell death in MCF-7 and HeLa cells, but inhibited the proliferation of these cells. In conclusion, DMH1 inhibits chemotherapeutic drug-induced autophagy response and the enhancement of efficacy of chemotherapeutic drugs by DMH1 is dependent on the cell sensitivity to drugs.

Keywords: 5-Fluorouracil; Autophagy; Cancer cells; Tamoxifen.

Graphical abstract

Figure 1

DMH1 potentiates the anticancer activity of CDDP for HeLa cells. (A) DMH1 inhibited CDDP-induced autophagy in HeLa cells. Cells were treated with DMH1 or CDDP for 24 h. *P<0.05 vs. Control; ^#P<0.05, ^##P<0.01 vs. CDDP. (B) Co-application of DMH1 enhanced the ability of CDDP to reduce HeLa cell viability. *P<0.05, **P<0.01 vs. Control; ^#P<0.05, ^##P<0.01 vs. counterpart CDDP dose. n=10.

Figure 2

DMH1 enhances the apoptotic induction effects of CDDP on HeLa cells after 24 h treatment. (A) DMH1 enhanced CDDP-induced cell death after 24 h treatment. The live cells were stained with calcein AM in green, and the dead cells were stained with ethidium homodimer-1 in red. (B) TUNEL staining showed that DMH1 enhanced CDDP-induced cell apoptosis after 24 h treatment. The analyzed cell number was 6219 in control group, 3468 in CDDP group and 3615 in CDDP plus DMH1 group. **P<0.01 vs. Control; ^##P<0.01 vs. CDDP.

Figure 3

DMH1 potentiates the anticancer activity of tamoxifen for MCF-7 cells. (A) DMH1 inhibited tamoxifen-induced autophagy in MCF-7 cells. Cells were treated with DMH1 or tamoxifen for 24 h. *P< 0.05 vs. Control; ^#P<0.05 vs. tamoxifen. (B) Co-application of DMH1 enhanced the ability of tamoxifen to reduce MCF-7 cell viability. *P<0.05, **P<0.01 vs. Control; ^#P<0.05, ^##P<0.01 vs. counterpart tamoxifen dose. n=10.

Figure 4

DMH1 inhibited 5-FU-induced autophagy in MCF-7 and HeLa cells but did not affect the inhibitory effects of 5-FU on MCF-7 and HeLa cell viability after 24, 48 and 72 h treatment. (A) DMH1 inhibited 5-FU-induced autophagy in HeLa cells. *P<0.05 vs. Control. ^#P<0.05, ^##P<0.01 vs. 5-FU. (B) DMH1(5 and 10 μmol/L) did not affect the inhibitory effects of 5-FU on HeLa cell viability after 24, 48 and 72 h treatment. **P<0.01 vs. Control. n=10. (C) DMH1 inhibited 5-FU-induced autophagy in MCF-7 cells. *P<0.05 vs. Control. ^#P<0.05 vs. 5-FU. (D) DMH1 (5 and 10 μmol/L) did not affect the inhibitory effects of 5-FU on MCF-7 cell viability after 24, 48 and 72 h treatment. **P<0.01 vs. Control. NS, no significance vs. counterpart drug dose. n=10.

Figure 5

DMH1 inhibits HeLa and MCF-7 cell proliferation. (A) DMH1 reduced HeLa cells viability. **P<0.01 vs. Control. n=10. (B) Representative photos of live and dead staining by which the live cells fluoresce green and death cells fluoresce red. (C) DMH1 reduced MCF-7 cells viability. *P<0.05, **P<0.01 vs. Control. n=10. (D) Representative photos of live- and dead staining by which the live cells fluoresce green and death cells fluoresce red. (E) DMH1 dose-dependently inhibited HeLa cell proliferation. *P<0.05, **P<0.01 vs. Control. n=4. (F) The time course of DMH1-inhibited cell proliferation in HeLa cells. **P<0.01 vs. Control. n=4.