YQ36: a novel bisindolylmaleimide analogue induces KB/VCR cell death

Abstract

Overexpression of multidrug resistance proteins P-glycoprotein (P-gp, MDR1) causes resistance of the tumor cells against a variety of chemotherapeutic agents. 3-(1-methyl-1H-indol-3-yl)-1-phenyl-4-(1-(3-(piperidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-3-yl)-1H-pyrrole-2,5-dione (YQ36) is a novel analogue of bisindolylmaleimide, which has been reported to overcome multidrug resistance. Here, we dedicated to investigate the anticancer activity of YQ36 on KB/VCR cells. The results revealed that YQ36 exhibited great antiproliferative activity on three parental cell lines and MDR1 overexpressed cell lines. Moreover, the hypersensitivity of YQ36 was confirmed on the base of great apoptosis induction and unaltered intracellular drug accumulation in KB/VCR cells. Further results suggested that YQ36 could not be considered as a substrate of P-gp, which contributed to its successfully escaping from the efflux mediated by P-gp. Interestingly, we observed that YQ36 could accumulate in nucleus and induce DNA damage. YQ36 could also induce the activation of caspase-3, imposing effects on the mitochondrial function. Collectively, our data demonstrated that YQ36 exhibited potent activities against MDR cells, inducing DNA damage and triggering subsequent apoptosis via mitochondrial pathway.

Figure 1

The structure of YQ36.

Figure 2

YQ36 inhibited the proliferation on three panels of chemosensitive tumor cells and their chemoresistance subline. (a) Human oral squamous carcinoma cell lines (KB and KB/VCR) were treated with YQ36 or DOX (0.16–100 μM) for 48 hours. Points represent the mean fractional survival; error bars represent standard deviation. (b) Human breast cancer cell lines (MCF-7 and MCF-7/DOR) were treated with YQ36 or DOX (0.16–100 μM) for 48 hours. Points represent the mean fractional survival; error bars represent standard deviation. (c) Human leukemia cell lines (K562 and K562/ADR) were treated with YQ36 or DOX (0.32–100 μM) for 48 hours. Points represent the mean fractional survival; error bars represent standard deviation.

Figure 3

The action of YQ36-induced antimultidrug-resistance on KB/VCR cells.

Figure 4

YQ36 caused apoptosis in KB/VCR cells in vitro. (a) YQ36 induced a characteristic fragmentation of DNA in KB/VCR cells (10 μM, 6–24 hours). (b) The result of measuring caspase-3/7 activity indicated YQ36 induced KB/VCR cells apoptosis in time-dependent manner. The error bars represent standard deviation and **represent P < .01 versus control. (c) Caspase activation and mitochondrial pathway involved in YQ36-induced apoptosis. Cells were treated with YQ36 (10 μM) for 6, 12, and 24 hours, and whole-cell lysates were collected and immunoblotted with indicated antibodies. (d) The expressions of antiapoptotic proteins were decreased in KB/VCR cells (10 μM YQ36, 6–24 hours). (e) The protein expression of Bax in mitochondrial fraction after treated with YQ36 (10 μM) for 6, 12, and 24 hours, and the HSP60 was used as a housekeeper for mitochondrial fraction. (f) Densitometric analysis of expression of Bcl-2 and Bax relative to the control.

Figure 5

YQ36-caused apoptosis was caspase-dependent. (a) The morphology of KB/VCR cells treated with YQ36 (10 μM, 48 hours) plus with or without caspase inhibitor (100 μM BOC-D-FMK and 40 μM Z-DEVD-FMK) (100×). (b) The survival rate of KB/VCR cells treated with YQ36 (10 μM, 48 hours) plus with or without caspase inhibitor (100 μM BOC-D-FMK and 40 μM Z-DEVD-FMK).

Figure 6

YQ36-mediated DNA damage in KB/VCR cells. (a) The YQ36 accumulated in nucleus was detected by fluorescent microscope and the arrow indicated that YQ36 accumulated in nucleus. (b) Upregulated p-p53 was detected in YQ36-induced DNA damage by Western Blotting.