Cancer Letters

Cancer Letters

Volume 415, 28 February 2018, Pages 73-85
Cancer Letters

Original Articles
Ginsenoside Rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of NF-κB mediated epithelial–mesenchymal transition and stemness

https://doi.org/10.1016/j.canlet.2017.11.037Get rights and content

Highlights

  • Hypoxia reduced cisplatin chemosensitivity in lung cancer cells.

  • Hypoxia induced EMT and stemness mediated by the NF-κB signaling pathway.

  • Rg3 inhibited NF-κB activation in hypoxia in a concentration/time-dependent manner.

  • Rg3 + cisplatin inhibited hypoxia-induced EMT and stemness in vitro and in vivo.

  • This is the first report that Rg3 had an anti-stemness effect.

Abstract

Cisplatin is a first line chemotherapy in lung cancer, but decreased susceptibility may limit its application. In solid tumors, hypoxia alters the microenvironment and is associated with proliferation, metastasis, and drug sensitivity. The hypoxia-induced desensitization of cisplatin is not clearly elucidated. 20 (R)-Ginsenoside (Rg3), the traditional Chinese medicine, is extracted from ginseng and has antitumor activities. In this study, we evaluated if Rg3 is effective in improving cisplatin sensitivity by blocking hypoxia. We found that the inhibition of proliferation potential by cisplatin was reduced in cobalt chloride (CoCl2)-induced hypoxia in lung cancer cells. Hypoxia caused alterations in epithelial–mesenchymal transition (EMT), which were detected by cellular morphology and EMT protein markers, and in stemness analyzed by spheroid formation and marker molecules. Hypoxia also activated EMT, which was mediated by the nuclear factor κB (NF-κB) pathway, and stemness, and Rg3 inhibited the activation of the NF-κB pathway. Furthermore, Rg3 could increase the sensitivity to cisplatin by inhibiting EMT and stemness in hypoxic lung cancer cells, and this effect was confirmed in vivo. In conclusion, Rg3 may improve the sensitivity of cisplatin in lung cancer therapy.

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].

References (45)

  • G.S. Oh

    Pharmacological activation of NQO1 increases NAD(+) levels and attenuates cisplatin-mediated acute kidney injury in mice

    Kidney Int.

    (2014)
  • S.M. Kim

    Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-kappaB

    Eur. J. Pharmacol.

    (2010)
  • C. Wohlkoenig

    Hypoxia-induced cisplatin resistance is reversible and growth rate independent in lung cancer cells

    Cancer Lett.

    (2011)
  • M. Zhao

    Hypoxia-induced cell stemness leads to drug resistance and poor prognosis in lung adenocarcinoma

    Lung Cancer

    (2015)
  • K.P. Mishra et al.

    Hypoxia modulates innate immune factors: a review

    Int. Immunopharmacol.

    (2015)
  • S. Junmin

    Ginsenoside Rg3 inhibits colon cancer cell migration by suppressing nuclear factor kappa B activity

    J. Tradit. Chin. Med.

    (2015)
  • L.A. Torre

    Global cancer statistics, 2012

    CA Cancer J. Clin.

    (2015)
  • B. Zaric

    Adjuvant chemotherapy and radiotherapy in the treatment of non-small cell lung cancer (NSCLC)

    J. Thorac. Dis.

    (2013)
  • A. Challapalli et al.

    Molecular mechanisms of hypoxia in cancer

    Clin. Transl. Imaging

    (2017)
  • J.P. Cosse et al.

    Tumour hypoxia affects the responsiveness of cancer cells to chemotherapy and promotes cancer progression

    Anticancer Agents Med. Chem.

    (2008)
  • C. Yip

    Molecular imaging of hypoxia in non-small-cell lung cancer

    Eur. J. Nucl. Med. Mol. Imaging

    (2015)
  • E. Giannoni et al.

    EMT and oxidative stress: a bidirectional interplay affecting tumor malignancy

    Antioxid. Redox Signal

    (2012)
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