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

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

Abstract and Introduction

Abstract

Aim: To explore whether triptolide (TPL) can enhance drug sensitivity of resistant myeloid leukemia cell lines through downregulation of HIF-1α and Nrf2. Materials & methods: HL60/A and K562/G cells were subjected to different treatments and thereafter an methyl thiazole tetrazolium bromide assay, flow cytometry, western blot and real-time PCR were used to determine IC₅₀, apoptotic status and expression of Nrf2, HIF-1α and their target genes. Results: Doxorubicin- or imatinib-induced apoptosis was enhanced when anticancer agents were used in combination with TPL. When combined with TPL, both doxorubicin and imatinib downregulate Nrf2 and HIF-1α expression at protein and mRNA levels. Genes downstream of Nrf2, for example, NQO1, GSR and HO-1, as well as target genes of HIF-1α, for example, BNIP3, VEGF and CAIX are also downregulated at the mRNA level. Conclusion: TPL is able to enhance drug sensitivity of resistant myeloid leukemia cell lines through downregulation of HIF-1α and Nrf2.

Introduction

Although therapies for leukemia, including chemotherapy and bone marrow transplantation, have been developing very fast in recent years, patients still suffer from relapse and treatment-related complication.^[1] It is well-known that the emergence of cancer cell resistance might be responsible for relapse while the addition of another cytotoxic drug into the chemotherapy regimen commonly leads to more serious side effects rather than improving the therapeutic index.^[1] Thus, it is a challenge to find a novel drug sensitizer that enhances specific cancer killing of conventional anticancer drugs without general toxicity to cancer patients.

Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii has been used in traditional Chinese medicine for centuries.^[2] It has been employed in the treatment of autoimmune diseases such as rheumatoid arthritis, nephritic syndrome, ulcerative colitis, asthma and idiopathic pulmonary fibrosis, as well as prevention of rejection in transplantation.^[3] Recently, TPL has also been shown to have strong anticancer properties both in vitro and in vivo. It could induce apoptosis in a variety of cancer cell lines such as leukemia,^[4,5] pancreatic cancer,^[6] adrenal cancer,^[7] cholangiocarcinoma^[8] and colorectal cancer.^[9] However, the clinical applications of TPL are limited by its narrow therapeutic window and severe toxicity in the digestive, reproductive, urogenital and blood circulatory systems.^[10] Yet, it has been reported that a relatively low dosage of TPL can enhance the cytotoxicity of some cytokines and conventional anticancer drugs, which indicates that TPL might be a promising chemotherapy sensitizer.^[11–13] Moreover, the mechanism of action of TPL in enhancing drug-induced apoptosis in cancer cells remains unclear. Establishing the mechanism might help to reassure patients with certain kind of genes who might be more sensitive to TPL treatment while providing a much more individualized therapy. This will help to define how an individual's genetic inheritance affects the body's response to TPL, which is in accordance with the idea of pharmacogenomics.

Nrf2 is a transcription factor that plays a vital role in activating an antioxidant response that decreases reactive oxygen species, detoxifies harmful chemicals and ultimately protects cancer cells from cytotoxic insults.^[14] Previous publications report that activation of Nrf2 by sulforaphane in wild-type fibroblasts can protect cells from the attack of many types of electrophiles whereas Nrf2-null cells are more sensitive to many of the same compounds, indicating the ability of Nrf2 to confer resistance to many xenobiotics.^[15] Previous publications also demonstrate a relationship between Nrf2 and chemoresistance.^[16,17] Moreover, Nrf2 might be controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK3 activity and thus activation of GSK3 overcomes Nrf2-mediated drug resistance by increasing Nrf2 turnover.^[18] So, targeting Nrf2 might be an effective way to induce cancer cell apoptosis.

HIF-1α is another critical transcription factor responsible for adaptive responses of cancer cells to reduced O₂ availability.^[19] It is extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis with elevated levels being found in various human primary and metastatic cancers.^[20,21] It is also important for the survival of leukemia stem cells, which are considered to be responsible for relapse.^[22] Moreover, increase of HIF-1α expression in leukemia patients is related to poor treatment outcome.^[23] Thus, HIF-1α has been proposed as a promising anticancer target.

Doxorubicin (DOX)^[24] and imatinib (IM)^[25] are two conventional anticancer drugs used in clinical settings. However, drug-resistance-induced leukemia recurrence after DOX- and IM-based therapy might lead to the death of patients. This study aimed to investigate whether TPL could enhance drug sensitivity of the DOX-resistant leukemia cell line HL60/A and the IM-resistant cell line K562/G to DOX and IM, and to elucidate the potential molecular mechanisms and thus provide an experimental basis for further more individualized therapy based on an individual's genetic inheritance.

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Table 1. Cytotoxicity of anticancer agents to leukemia cells *in vitro*.
Cell type	IC₅₀ (µM)	Fold resistance	p-value
HL60	0.28 ± 0.02	78.68	<0.05
HL60/A	22.03 ± 0.22
K562	2.28 ± 0.15	68.56	<0.05
K562/G	156.30 ± 4.06

HL60 and HL60/A cells were exposed to doxorubicin and K562 and K562/G cells were exposed to imatinib. After 24 h of drug exposure the cytotoxicity and IC₅₀ values were determined by methyl thiazole tetrazolium bromide assay.
Values are expressed as mean ± standard deviation of three independent experiments.

Table 2. Triptolide enhances the cytotoxicity of anticancer agents to leukemia cells *in vitro*.
Cell type	Treatment	IC₅₀ (µM)	Reversal fold	p-value
HL60/A	DOX	14.36 ± 2.23	1.82	<0.01
	TPL + DOX	7.90 ± 0.33
K562/G	IM	62.54 ± 4.33	1.18	<0.05
	TPL + IM	53.11 ± 0.57

HL60/A cells were exposed to DOX with or without TPL (14 nM) for 48 h in vitro with IC₅₀ detected by methyl thiazole tetrazolium bromide assay while K562 and K562/G cells were exposed to IM with or without TPL (25 nM) for 48 h.
Values are expressed as mean ± standard deviation of three independent experiments.
DOX: Doxorubicin; IM: Imatinib; TPL: Triptolide.

Table 3. Triptolide enhanced anticancer agents induced leukemia cell apoptosis.
Cell type	Control	TPL	Agent^†	Agent + TPL	p-value
HL60/A (%)	5.28 ± 0.80	7.09 ± 0.46	19.55 ± 1.70	72.62 ± 4.83	<0.01
K562/G (%)	5.88 ± 0.40	7.90 ± 0.59	24.78 ± 1.12	77.52 ± 7.75	<0.01

HL60/A and K562/G cells were exposed to different treatments for 24 h. The apoptotic ratios were detected using flow cytometry. Values are expressed as mean ± standard deviation of three independent experiments. TPL was administered as follows: HL60/A: 14 nM; K562/G: 25 nM.
^†HL60/A: doxorubicin (7μM); K562/G: imatinib (50μM).
TPL: Triptolide.

Sidebar

Executive Summary

Background

Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in traditional Chinese medicine for centuries. Its usage is limited by its narrow therapeutic window.
Previous reports have shown TPL could be used as a chemosensitizer.
Methods
The mRNA levels of Nrf2 and HIF-1α of HL60, HL60/A, K562 and K562/G were determined using real-time PCR.
Cytotoxicity of TPL to HL60, HL60/A, K562 and K562/G was determined using an methyl thiazole tetrazolium bromide (MTT) assay.
HL60/A and K562/G cells were subjected to different treatments and thereafter an MTT assay, flow cytometry, western blot and real-time PCR were used to determine IC ₅₀, apoptotic status and expression of Nrf2, HIF-1α and their target genes.
Results
TPL could enhance the drug sensitivity of resistant myeloid leukemia cell lines
- TPL enhances the drug sensitivity of HL60/A cells to doxorubicin (DOX; IC ₅₀ : 14.36 ± 2.23 vs 7.90 ± 0.33 µM; 1.82-fold; p < 0.01) and K562/G cells to imatinib (IM; IC ₅₀ : 62.54 ± 4.33 vs 53.11 ± 0.57 µM; 1.18-fold; p < 0.05).
TPL could enhance anticancer agent-induced apoptosis
- Apoptosis induced by DOX or IM is enhanced when used in combination with TPL in HL60/A or K562/G cells (HL60/A: 19.55 ± 1.70% vs 72.62 ± 4.83%, p < 0.01; K562/G: 24.78 ± 1.12% vs 77.52 ± 7.75%, p < 0.01).
TPL, in combination with anticancer agents, downregulates Nrf2 and its target genes
- Nrf2 is an important protective factor that is correlated with chemoresistance in cancer cells.
- In combination with TPL, both DOX and IM downregulate Nrf2 expression in HL60/A and K562/G cells at both protein and mRNA levels. Genes downstream of Nrf2, for example, NQO1, GSR and HO-1 were also downregulated at the mRNA level.
TPL, in combination with anticancer agents, downregulates HIF-1α and its target genes
- HIF-1α is the major mediator of hypoxic responses in malignant cells. High expression of it is correlated with poor clinical outcome in patients. It is also important for the survival of leukemia stem cells, which are responsible for relapse.
- In combination with TPL, both DOX and IM downregulate HIF-1α expression in HL60/A and K562/G cells at both the protein and mRNA levels. Genes downstream of HIF-1α such as BNIP3, VEGF and CAIX are also downregulated by DOX or IM combined with TPL in HL60/A or K562/G cells at the mRNA level.
Conclusion
Our study demonstrates that TPL could enhance drug sensitivity of HL60/A and K562/G in vitro through downregulation of Nrf2 and HIF-1α.
This work indicates a new role for TPL in cancer therapeutics. Further studies may lead to it being used as a chemosensitizer for clinical use in leukemia treatment.
Our study also provides an experimental basis for further study focusing on the ability of TPL in enhancing drug sensitivity in patients with certain kinds of genes to promote more individualized therapy.
However, this is just a preliminary exploration, which needs further study focusing on chronic phase cells from primary chronic myeloid leukemia tissue and primary acute myeloid leukemia tissue while exploring the ability of TPL in enhancing the cytotoxicity of other first-line drugs for leukemia, such as daunorubicin and cytarabine.