Translated Abstract
Background:
Gastric cancer is a common malignancy and its prognosis is still dismal in most part of the world. At present, surgical resection is still the main therapeutic strategy for gastric cancer, supplemented with perioperative chemotherapy, chemoradiotherapy and/or immunotherapy. Accumulated evidence has demonstrated the efficacy of adjuvant chemoradiotherapy for promoting survival and local control for gastric cancer patients and radiotherapy has got its recognized role in the management of advanced gastric cancers with high risk. However, radioresistance, as well as the acute and late toxicity such as late renal toxicity, endocrine pancreatic insufficiency and reduction of quality of life resulting from radiotherapy, have limited its use in clinical practice. Therefore, it is urgent and important for us to search an effective radiosensitizer so as to improve the efficacy of radiotherapy and to reduce the toxicity to surrounding tissues and organs of radiation field.
The radiosensitivity of cancer cells depends on several aspects, such as apoptosis sensitivity, cell cycle distribution of irradiated cells, the level of multiple oncogenic or tumor suppressing genes, the repair of sublethal DNA damage and the signaling pathways affected by ionizing radiation (IR). β-Elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane) is a novel anti-cancer agent extracted from the Chinese medicinal herb Curcuma wenyujin. Our group and others have found its anti-cancer potential in a variety of cancer cells. The involved mechanisms include inhibition of multiple oncogenic signaling pathways, such as ERK and PI3K/Akt signaling, and thus inducing apoptosis and cell cycle arrest. These signaling pathways and cytological behaviors have been demonstrated to be critical for regulating the radiosensitivity of cancer cells. Thus we assumed that β-elemene could enhance radiosensitivity of gastric cancer cells through multiple mechanisms.
Objective:
The current study aims to explore the potential of β-elemene as a radiosensitizer for gastric cancer cells and the underlying mechanisms, and to investigate the possibility of β-elemene as a radiosensitizer in clinical practice.
Methods:
1. SGC7901, MKN45, MKN28, N87 and AGS human gastric cancer cell lines were used to screen for radioresistant gastric cancer cell lines. A 3-(4,5-dimeth-ylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) assay was used to determine the effects of β-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines and to choose doses of drug used in subsequent studies. Clonogenic survival assay and Annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death, respectively.
2. A proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), was employed to screen the proteins regulated by β-elemene pretreatment prior to IR in SGC7901 gastric cancer cell line. Protein levels of p21-activated protein kinase-interacting protein 1(PAK1IP1), Bcl-2-associated transcription factor 1 (BTF) and topoisomerase 2-alpha (TOPIIα) were validated by western blots. After evaluation of the proteomic result, assistant with previous studies, for the assessment of potential underlying molecules for radiosensitization effect of β-elemene, protein levels of PAK1IP1, total PAK1 (t-PAK1) (p21-activated protein kinase 1), phospho-PAK1 (T423), phospho-ERK1/2 (Thr202/Tyr204) and cleaved Caspase-3 (17 kDa) were assessed by western bolts.
3. IPA-3 was used as a specific small molecule inhibitor of PAK1 to target PAK1 signaling. A MTT assay was used to determine the effects of β-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines. Clonogenic survival assay and Annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death altered by IPA-3 prior to irradiation, respectively. Protein levels of PAK1IP1, total PAK1 (t-PAK1), phospho-PAK1 (T423), phospho-ERK1/2 (Thr202/Tyr204) and cleaved Caspase-3 (17 kDa) were assessed by western bolts.
Results:
1. MKN45 and SGC7901 gastric cancer cell lines were relatively more resistant to IR. β-Elemene inhibited viability of MKN45 and SGC7901 gastric cancer cells in a dose-dependent manner. β-Elemene pretreatment decreased clonogenic survival following IR in MKN45 and SGC7901 gastric cancer cell lines. Additionally, β-elemene pretreatment prior to IR increased radiation-induced cell death compared with IR alone in MKN45 (10.4±0.9% vs. 34.8±2.8%, P＜0.05) and SGC7901 (11.6±0.9% vs. 46.7±5.2%, P＜0.05) human gastric cancer cell lines, respectively, consistent with the level of cleaved Caspase-3 (17 kDa).
2. Through iTRAQ analysis, 147 up-regulated proteins and 86 down-regulated proteins were identified in response to β-elemene treatment in SGC7901 gastric cancer cells. Western blot validation showed that PAK1IP1 and BTF were up-regulated and TOPIIα was down-regulated. The trends closely match the iTRAQ proteomic results.
3. We found that β-elemene up-regulated PAK1IP1 and down-regulated phospho-PAK1 (T423) and phospho-ERK1/2 in a dose-dependent manner in SGC7901 gastric cancer cells. IR increased the level of phospho-PAK1 (T423). Compared with IR alone, β-elemene pretreatment prior to IR increased the level of PAK1IP1, decreased radiation-induced PAK1 and ERK1/2 phosphorylation in SGC7901 gastric cancer cells.
4. IPA-3 suppressed the viability of gastric cancer cells in a dose- and time-dependent manner. Inhibition of PAK1 using IPA-3 decreased clonogenic survival following IR. In addition, IPA-3 increased radiation-induced cell death in MKN45 (13.4±0.3% vs. 26.6±1.0%, P＜0.05) and SGC7901 (16.0±0.6% vs. 37.3±1.7%, P＜0.05) gastric cancer cell lines, respectively, consistent with the level of cleaved Caspase-3 (17 kDa). Western blot results showed that IPA-3 decreased radiation-induced PAK1 and ERK1/2 phosphorylation.
Conclusions:
1. β-Elemene enhances radiosensitivity of gastric cancer cells and the mechanism involves inhibition of PAK1 signaling.
2. PAK1 can be activated by ionizing radiation and mediates radioresistance of gastric cancer cells. Targeting PAK1 signaling is a means to sensitize gastric cancer cells to ionizing radiation.
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