Original ArticlesGinsenoside Rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of NF-κB mediated epithelial–mesenchymal transition and stemness
Introduction
Lung cancer is the leading cause of cancer-related death in the world [1], [2]. There are two types, small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC is the most common, and accounts for 85% of lung cancers [3]. Although surgery is the most effective treatment for NSCLC, chemotherapy is also an option and is an important adjuvant therapy for patients after surgery [4]. Though many new antitumor regimens are used for NSCLC, platinum-based chemotherapy remains the first-line treatment [5]. Cisplatin is still one of the most potent platinum agents, displaying antitumor activities against a wide variety of solid tumors, including lung, bladder, and ovarian cancers [6]. However, clinical applications of cisplatin are limited because of reduced chemosensitivity and toxicity. Therefore, there is an urgent need to elucidate the molecular mechanism of cisplatin desensitization, and search for more effective and less toxic drugs to be combined with cisplatin.
Hypoxia is the most prominent sign of a tumor microenvironment and results from tumor cell proliferation that is faster than angiogenesis or abnormal new blood vessels [7], [8]. Due to the imperfect structure of new blood vessels and irregular enlargement of the vascular lumen, vessels can easily collapse, leading to chronic perfusion insufficiency and temporary acute hypoxia. This results in necrosis or apoptosis of tumor cells. However, there are still some cells that can tolerate hypoxia, escape from hypoxia-induced apoptosis or death, and exhibit more malignant biological phenotypes and stronger invasion and metastasis [9]. Hypoxia commonly exists in a variety of solid tumors, and is closely associated with tumor proliferation and metastasis, clinical stage, therapeutic efficacy, as well as the prognosis of the patients [10]. Therefore, the hypoxic microenvironment has become a focus in cancer research and treatment. The accumulated data suggest that hypoxia induces characteristic alterations in molecular expression and cellular morphology. Hypoxia induced factor-1α (HIF-1α) is the active form of HIF-1. It is highly expressed in hypoxic solid tumors, and used as an indicator or a marker to assess the degree of hypoxia and malignancy [11]. Elevated HIF-1α is found in many cancer cells with cobalt chloride (CoCl2)-induced hypoxia. Studies also revealed that hypoxia can promote epithelial–mesenchymal transition (EMT) and cancer cell stemness, which facilitates survival. The EMT is considered the driving force of tumor progression. During EMT, the mesenchymal markers N-cadherin and Vimentin are upregulated; while, the expression of epithelial marker E-cadherin is downregulated and transcriptionally repressed by Snail [12]. The tumor cells also undergo a stemness transformation in hypoxia [13], which induces the potential for self-renewal [14]. The stemness marker molecules include the transcription factors SOX2, NANOG, and OCT4 and the surface marker CD44 [15]. Hypoxic transformations of the cancer cells are medicated by the activated NF-κB signaling pathway [16]. The hypoxia-induced changes in tumor cells mentioned above are intrinsically associated with decreased chemosensitivity, e.g., cisplatin desensitization. Thus, inhibiting hypoxia may be a promising anticancer strategy for lung cancer [17].
The compound, 20 (R)-Ginsenoside Rg3 (Rg3), is an active monomer extracted from the traditional Chinese medicine ginseng [18], [19]. It was approved by the Chinese Food and Drug Administration as a cancer therapy in 2000, and was listed as the designated medicine for treating NSCLC in the National Comprehensive Cancer Network clinical practice guidelines (Chinese version) in 2006 and 2007. Rg3 has multiple antitumor effects, including inhibition of proliferation, metastasis, and angiogenesis; induction of apoptosis; elevation of chemotherapeutic susceptibility; and immune stimulation [20]. The suppression of hypoxia by Rg3 is rarely reported. Combinations of Rg3 and chemotherapeutic drugs have improved treatment efficacy, and could reduce the toxicity of chemotherapy [21], [22], [23]. Here, we hypothesized that Rg3 could inhibit hypoxia, and increases the sensitivity to cisplatin. We found that hypoxia increased the proliferation of lung cancer cells, and decreased the sensitivity to cisplatin in CoCl2-induced hypoxic cells; while, Rg3 inhibited EMT and stemness by inactivating the NF-κB signaling pathway. In hypoxia, Rg3 + cisplatin improved chemotherapy sensitivity in lung cancer both in vitro and in vivo.
Section snippets
Cell culture
Human NSCLC cell lines (SPC-A1, H1299 and A549) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in humidified air containing 5% CO2.
Reagents
The Rg3 was provided by the Dalian Fusheng Pharmaceutical Company (Dalian, China). Cisplatin and CoCl2 were obtained from Sigma Chemical Inc. (St. Louis, Missouri, USA). Rg3 was dissolved in serum-free
Hypoxia decreases the sensitivity of human NSCLC cells to cisplatin
Different concentrations of CoCl2 (0, 100, 200, 400 μM) were used to induce hypoxia in three different kinds of NSCLC cells (Fig. 1A). Hypoxia was also induced in the three different NSCLC cells at different times (0, 12, 24, and 48 h) using 200 μM of CoCl2 (Fig. 1A). Our results indicated that HIF-1α expression increased over 48 h after treatment with 200 μM CoCl2. On the basis of this result, we used 200 μM of CoCl2 to induce hypoxia in this study. Cisplatin drug sensitivity in NSCLC cells
Discussion
Cisplatin is widely used as a clinical chemotherapy, but its toxicity and desensitization are concerns [24]. Ototoxicity and nephrotoxicity are prominent with cisplatin cancer treatment e.g., high concentrations of cisplatin combined with radiotherapy caused irreversible hearing loss, and about one third of patients developed acute kidney injuries [25], [26], [27], [28]. Although lowering the cisplatin dosage reduces the toxicity, its antitumor efficacy is also weakened. Therefore, a small dose
Conflicts of interest statement
The authors declare that they have no competing interests.
Acknowledgements
The project was supported by a National Natural Science Foundation of China Research Grant [NO:81572881, NO:81602508].
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These authors made equal contributions as first author.