Triptolide, a Chinese Herbal Extract, Enhances Drug Sensitivity of Resistant Myeloid Leukemia Cell Lines Through Downregulation of HIF-1α and Nrf2

Feili Chen; Yuejian Liu; Shiyun Wang; Xutao Guo; Pengcheng Shi; Weiguang Wang; Bing Xu

Disclosures

Pharmacogenomics. 2013;14(11):1305-1317. 

In This Article

Results

Resistant Cell Lines Express More HIF-1α & Nrf2 Than Their Parental Cell Lines

To further confirm the important role of HIF-1 α and Nrf2 in the drug resistance of HL60/A and K562/G, mRNA levels of these two genes were detected using an RT-PCR method. Results showed that resistant cell lines have higher mRNA levels of HIF-1α and Nrf2 than their parental cell lines (Figure 1).

Figure 1.

Resistant cell lines have higher HIF-1α and Nrf2 mRNA levels than parental cell lines.
Values are expressed as mean ± standard deviation of three independent experiments.
*p < 0.05.

Cytotoxicity of the Individual Anticancer Agents in Leukemia Cell Lines

Cytotoxic effect of DOX, IM and TPL on HL60, HL60/A, K562 and K562/G cell lines was determined by an MTT assay. The drug-resistant cell lines, HL60/A and K562/G, were developed by exposing the parental cell lines (HL60 and K562) to increasing concentrations of DOX or IM as previously reported.[26,27] In comparison with the parental HL60 cells (IC50 = 0.28 ± 0.02 µM), the HL60/A cells are 78.68-fold resistant (IC50 = 22.03 ± 0.22 µM, p < 0.05) to DOX (Table 1). Similarly, the K562/G cells are 68.56-fold resistant (IC50 = 156.30 ± 4.06 µM, p < 0.05) to IM when compared with the parental K562 cells (IC50 = 2.28 ± 0.15 µM; Table 1). Furthermore, the cytotoxic effect of TPL on HL60/A and K562/G cell lines, as well as their parental cell lines HL60 and K562, was assayed using an MTT assay after exposure to a serial concentration of TPL for 48 h. TPL demonstrates a high toxicity to all cell lines. Resistant cell lines are slightly less sensitive to TPL than parental ones (IC50-HL60: 34.44 ± 2.81 nM vs IC50-HL60/A: 43.06 ± 1.06 nM, p < 0.05; IC50-K562: 46.68 ± 3.48nM vs IC50-K562/G: 55.14 ± 5.22nM, p < 0.05; Figure 2A & D).

Figure 2.

Cytotoxicity of drugs in leukemia cell lines detected by methyl thiazole tetrazolium bromide assay.
(A) HL60/A or HL60 cells were exposed to different concentrations of TPL for 48 h. (B) HL60/A cells were exposed to different concentrations of DOX with or without TPL (14 nM) for 48 h. (C) Synergy was quantified by combination index analysis and expressed as combination index versus fraction affected. (D) K562/G and K562 cells were exposed to different concentrations of TPL for 48 h. (E) K562/G cells were exposed to different concentrations of IM with or without TPL (25 nM) for 48 h. (F) Synergy was quantified by combination index analysis and expressed as combination index versus fraction affected. Values are expressed as mean ± standard deviation of three independent experiments.
p > 0.05; *p < 0.05; **p < 0.01.
DOX: Doxorubicin; IM: Imatinib; TPL: Triptolide.

Enhancing the Effect of TPL on the Cytotoxicity of Anticancer Agents to Leukemia Cell Lines

To determine the ability of TPL to enhance sensitivity to drugs, HL60/A and K562/G cells were exposed to increasing concentrations of DOX or IM, respectively, with TPL at a constant IC20 concentration (14 nM and 25 nM, respectively) for 48 h. The cytotoxicity effect of DOX and DOX combined with TPL on HL60/A cells are presented in Figure 2B & Table 2, while the effect of IM and IM combined with TPL on K562/G are presented in Figure 2E & Table 2. In comparison with anticancer agent alone, TPL enhances the cytotoxicity of DOX (IC50: 14.36 ± 2.23 vs 7.90 ± 0.33 µM, 1.82-fold; p < 0.01) to HL60/A and IM (IC50: 62.54 ± 4.33 vs 53.11 ± 0.57 µM, 1.18-fold; p < 0.05) to K562/G. CI was also analyzed and the results are presented in Figure 2C & F. Results of CI indicate that TPL and anticancer agents have synergistic effects. The fraction affected was below 60% (DOX) and 90% (IM).

TPL Enhanced Anticancer Agents Induced Leukemia Cell Apoptosis

To determine the combination effect of TPL and anticancer agents, HL60/A and K562/G cells were exposed to DOX (7 µM) or IM (50 µM) with or without TPL (14 nM and 25 nM, respectively) for 24 h. Apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Apoptotic ratio of cells treated with DOX or IM together with TPL significantly increased compared with cells treated with DOX or IM alone (DOX: 19.55 ± 1.70% vs 72.62 ± 4.83%, p < 0.01; IM: 24.78 ± 1.12% vs 77.52 ± 7.75%, p < 0.01, Figure 3 & Table 3).

Figure 3.

Apoptotic ratio of leukemia cells was higher in triptolide combined with anticancer agent group than that in anticancer agent group.
Apoptotic ratio is determined as the following: first, cells were stained with PI and fluoresceinated anti-annexin V (annexin-V) antibody and then cells were subjected to flow cytometry analysis. Histograms represent the total percentage of apoptotic cells (sum of the percentages of annexin V-positive/PI-negative, double-positive cells). (A & B) HL60/A cells were exposed to different treatments (DOX: 7 µM; TPL: 14 nM) for 24 h and then apoptotic ratios were determined using flow cytometry. Histogram shows the total percentage of apoptotic HL60/A cells in different treatment groups. (C & D) K562/G cells were exposed to different treatments (IM: 50 µM; TPL: 25 nM) for 24 h and then apoptotic ratios were determined using flow cytometry. The histogram shows the total percentage of apoptotic K562/G cells in different treatment groups.
p > 0.05; *p < 0.05; **p < 0.01.
DOX: Doxorubicin; IM: Imatinib; PI: Propidium iodide; TPL: Triptolide.

Combined use of TPL & Anticancer Agents Decreased Nrf2 Expression in Leukemia Cell Lines

A previous publication has demonstrated that downregulation of Nrf2 might sensitize cancer cells to chemotherapy drugs.[29] To explore whether decrease of Nrf2 expression in leukemia cell lines might contribute to the ability of TPL in enhancing the cytotoxicity of anticancer agents, Nrf2 expression was detected by western blotting and RT-PCR while mRNA levels of downstream genes of Nrf2 were also assessed using RT-PCR in HL60/A and K562/G cells treated with different drugs (DOX 7 µM plus TPL 14 nM; IM50 µM plus TPL 25 nM, respectively). Western blotting results shows that Nrf2 expression decreases in treated groups especially in cells treated with anticancer agents plus TPL (Figure 4). NQO1, GSR and HO-1 are downstream genes regulated by Nrf2. Our RT-PCR results showed Nrf2, NQO1, GSR and HO-1 decreased in all treated groups while the largest scale of decrease occurred in cells exposed to anticancer agents plus TPL (Figure 5).

Figure 4.

Western blotting and densitometry analysis.
TPL downregulated Nrf2 and HIF-1α expression detected by western blotting assay (A & C). Densitometry analysis of expression level from western blot (B & D). (A & B) HL60/A cells were exposed to different treatments as indicated (DOX: 7 µM; TPL: 14 nM). (C & D) K562/G cells were exposed to different treatments as indicated (IM: 50 µM; TPL: 25 nM). Values are expressed as mean ± standard deviation of three independent experiments.
p > 0.05; *p < 0.05; **p < 0.01.
DOX: Doxorubicin; IM: Imatinib; TPL: Triptolide.

Figure 5.

Triptolide downregulated Nrf2 and HIF-1α as well as target genes detected by RT-PCR.
(A & B) HL60/A cells were exposed to different treatments as indicated (DOX: 7 µM; TPL: 14 nM). (C & D) K562/G cells were exposed to different treatments as indicated (IM: 50 µM; TPL: 25 nM). Values are expressed as mean ± standard deviation of three independent experiments.
p > 0.05; *p < 0.05; **p < 0.01.
DOX: Doxorubicin; IM: Imatinib; TPL: Triptolide.

Combined use of TPL & Anticancer Agents Decreased HIF-1α Expression in Leukemia Cell Lines

Previous studies have shown downregulation of HIF-1α contributed to the anticancer properties of glucosamine hydrochloride.[30] Thus, whether anticancer agents plus TPL could alter the expression of HIF-1α was investigated. HL60/A and K562/G cells were exposed to different drug combinations (DOX 7 µM plus TPL 14nM; IM50 µM plus TPL 25 nM) for 24 h. The protein and mRNA extracted from the treated cells were then subjected to western blotting and RT-PCR analysis. Results show that HIF-1α is significantly downregulated in treated groups especially in the anticancer agents plus TPL group both at the protein and mRNA levels (Figures 4 & 5). The downstream genes of HIF-1α, for example, BNIP3, VEGF and CAIX are also downregulated at the mRNA level as shown in Figure 5 and CXCR4 at protein level as shown in Figure 4.

processing....