Effect Of Chlorin E6/SDT And Paclitaxel On The Proliferation Of Human Lung Adenocarcinoma Cell SPCA-1

Non small cell lung cancer is the leading cause of cancer death in the world. At the time of diagnosis,70%of patients are in advanced stages (stageⅢB or stageⅣ). For these patients, the combination of third-generation chemotherapy drugs with platinum is the standard first-line treatment. However, the major problem is that the efficacy of chemotherapy has reach a plateau, with 25%～35%of efficiency and 8-10 months of median survival time. Although targeted therapies of epidermal growth factor receptor (EGFR) and its cell signaling pathway have been used for lung caner, their efficiency is dependent on the state of EGFR. Moreover, the lifetime only can be prolonged 1-3 months in patients treated with chemotherapy in combination with anti-angiogenesis drug. How to improve the therapeutic effect and prolong the survival time of patients with NSCLC in the advanced stages is still a big problem in front of us.Sonodynamic therapy (SDT) is a promising new approach for cancer therapy. As photodynamic therapy (PDT) (activating a tumor-localizing photosensitizer with light), SDT functions by activating a tumor-localizing sonosensitizing agent with ultrasound, which generates reactive oxygen species. Compared with the light, ultrasound can penetrate and focus on the deep tissue, which makes sonodynamic therapy become a highly potential approach for cancer therapy. Due to most photosensitizers simultaneously having sonodynamic effect, photosensitizers are usually applied as sonosensitizer for sonodynamic research. Howeve there are two problems existed. The first is that the speed of those photosensitizers is slower whenever focusing on tumor tissue or clearing from normal tissue which induces higher phototoxic side effect. The second is how to enhance the efficacy of sonodynamic therapy through the combination with other treatmentChlorin e6 is the degradation products of chlorophyll. It is similar to hematoporphyrin(HpD)in structure. Previous studies have shown that it can highly selectively focus on tumor tissues, clear quickly from normal tissues and lower phototoxic side effects. As HpD, Chlorin e6 can be activated by light, producing photodynamic effect. Initially, the photodynamic effect of chlorin e6 was used to treat ehrlich ascites carcinoma cells and effectively inhibited the proliferation of the ehrlich ascites carcinoma cells. Its photodynamic effect against tumor was further confirmed in animal experiment for the treatment of squamous cell carcinoma. Recently, the photodynamic therapy of Chlorin e6 has been used in phase I clinical trial for the treatment of melanoma. Due to most photosensitizer simultaneously having sonodynamic effect, the first purpose of this study is to observe the SDT effect of Chlorin e6 on the proliferation of human lung adenocacinoma cell SPCA-1. Since pre-clinical and clinical experiments have confirmed that paclitaxel can enhance the radiosensitizer and photodynamic effect, the second purpose of this study is to explore whether paclitaxel can enhance the sonodynamic effect of chlorin e6 on the proliferation of SPCA-1 cell. PartⅠThe Sonodynamic Effect of Chlorin e6 on the proliferation of human lung adenocarcinoma cell SPCA-1Objective:To observe the acute cytotoxic effect of sonodynamic therapy (SDT) with Chlorin e6 on the proliferation of human lung adenocacinoma cell SPCA-1.Method:The cell viabilitys of SPCA-1 and normal peripheral mononuclear cell (PMNC) which were treated by ultrasonic irradiation and Chlorin e6 respectively, was detected by MTT assay. After the parameter of ultrasound irradiation(1.0W/cm2×30 s) and dosage of Chlorin e6 (0.05 mg/mL～0.2 mg/mL) were determined, the acute cytotoxic effect of SDT with Chlorin e6 on the proliferation of SPCA-1 and PMNC cells was evaluated by MTT assay and the morphological changes of SPCA-1 cells were observed using inverted microscope. Data are presented as mean±S.E.M. Experiments between two groups were analyzed by t test. Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.Results:1Effects of ultrasound and Chlorin e6 on the proliferation of SPCA-1 and PMNC cells 1.0 MHz ultrasound (1.0W/cm2-2.0W/cm2x60s) inhibited the cell proliferation of both SPCA-1 and PMNC cells in a intensity-dependent. The intensity of the 50%cell viability of both SPCA-1 and PMNC cells was 1.45 W/cm2and 1.44 W/cm2 respectively.There was no significant difference between the groups (P>0.05). Chlorin e6 (0.4mg/mL～3.2mg/mL) inhibited the cell proliferation of both SPCA-1 and PMNC cells in a dose-dependent manner respectively. The dosage of the 50%cell viability of both SPCA-1 and PMNC cells was 0.79 mg/mL and 0.97 mg/mL respectively. There was also no significant difference between the groups (P>0.05).2,The sonodynamic effects of ultrasound with chlorin e6 on the proliferation of SPCA-1 and PMNC cells The survival rates of both SPCA-1 and PMNC cells treated by 1.0 W/cm2 ultrasound was 79.6% and 78.0% respectively. Compared with the control group, there were both significant difference (P＜0.05), but no significant difference were found between SPCA-1 and PMNC cells (P>0.05).0.05 mg/mL,0.1 mg/mL and 0.2 mg/mL of chlorin e6 alone had no influence on the survival rates of both SPCA-1 and PMNC cells (P>0.05), but the survival rates of both SPCA-1 and PMNC cells exposed to ultrasound in combination with 0.05 mg/mL,0.1 mg/mL and 0.2 mg/mL of chlorin e6 were 66.3%,52.9%,28.3%and 78.7%,80.4%,75.3% respectively. Compared with the ultrasound (1.0W/cm2×60s) or Chlorin e6 (0.05 mg/mL～0.2 mg/mL) alone, There was significant difference in SPCA-1 cells (P< 0.05), not in PMNC cells (P>0.05).3,Morphological analysis of SPCA-1 cell survival induced by ultrasound and chlorin e6 alone or combination treatment Under high power microscopic view, the cells of round, lucent, integrated cell membrane and rich cytoplasm were considered as living cells, whereas the cells of polymorphic,cytomembrane ruptured and cytoplasm loss were considered as dead cells. The mortality of the control,1.0 W/cm2 ultrasound,0.2 mg/mL chlorin e6 and SDT(1.0 W/cm2 ultrasound with0.2 mg/mL chlorin e6) group were 1.6%,21.5%,2.3%and 69.8%, respectively. Compared with the ultrasound (1.0W/cm2×60s) or chlorin e6 (0.2mg/mL) alone, the combination treatment of ultrasound with Chlorin e6 induced more necrotic cells in SPCA-1 cells (P＜0.05).Conclusion:Results of the present study demonstrated that there was a significant selectively inhibitory effect of sonodynamic effect with Chlorin e6 on the SPCA-1 cell growth. Chlorin e6 may be a promising sonosensitizing agent for the treatment of non-small cell lung cancer. PartⅡPaclitaxel enhances the sonodynamic effect of chlorin e6 on the proliferation of human lung adenocarcinoma cell SPCA-1Objective:To observe the long persistence effect of SDT with Chlorin e6 on proliferation of human lung adenocarcinoma cell SPCA-1 and explore whether paclitaxel enhances the sonodynamic effect of chlorin e6 on the proliferation of SPCA-1 cell.Method:The cell viability of SPCA-1 cell, which were treated by ultrasonic irradiation, Chlorin e6 and paclitaxel respectively, was detected by MTT assay. After the parameter of ultrasound irradiation (1.0W/cm2×30 s) and dosage of Chlorin e6 (0.025 mg/mL) and paclitaxel (1×10-7 M) were determined, the cytotoxic effect of ultrasound/Chlorin e6 with or without paclitaxel respectively on the proliferation of SPCA-1 cell was evaluated by MTT assay. Data are presented as mean±S.E.M. Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.Results:1,Effects of ultrasound,chlorin e6 and paclitaxel on the proliferation of SPCA-1 cells 1.0 MHz ultrasound, at the intensity of 1.0W/cm2～2.0W/cm2×60s, inhibited the cell proliferation of SPCA-1 cells in a intensity-dependent manner. Both Chlorin e6 and paclitaxel inhibited the proliferation of SPCA-1 cells in a dose-dependent manner at the concentration of 0.05mg/mL～1.6mg/mL and 1×10-9 M～1×10-6 M respectively.2,The sonodynamic effect of chlorin e6 on the proliferation of SPCA-1 cells The survival rates of SPCA-1 cells treated by 1.0 W/cm2 ultrasound was 78.8%. Compared with the control group, there was significant difference (P＜0.05). whereas the cell viability of SPCA-1 cells treated with 0.025 mg/mL Chlorin e6 was 95.0%, Compared with the control group, there was no significant difference (P>0.05). When SPCA-1 cells were exposed to 1.0 W/cm2 ultrasound in combination with 0.025 mg/mL Chlorin e6, the survival rates was 56.9%. Compared with the ultrasound (1.0 W/cm2×60s) or Chlorin e6 (0.025 mg/mL) alone, the inhibitory effect on the proliferation of SPCA-1 cells was significantly enhanced by the combination of ultrasound with chlorin e6 (P< 0.05).3,The sonodynamic effect of chlorin e6 with or-withoutpaclitaxel on the. proliferation of SPCA-1 cells respectively The survival rates of SPCA-1 cells treated by 1.0 W/cm2 ultrasound combined with 0.025 mg/mL Chlorin e6 was 56.9%. Compared with the control group, there was significant difference (P＜0.05). the cell viability of SPCA-1 cells treated with 1×10-7 M paclitaxel was 54.6%, Compared with the control group, there was also significant difference (P＜0.05). When SPCA-1 cells were exposed to 1.0 W/cm2 ultrasound plus 0.025 mg/mL Chlorin e6 in combination with 1×10-7 M paclitaxel, the survival rates was 8.5%. Compared with paclitaxel (1×10-7M) or sonodynamic effect (1.0 W/cm2×60s ultrasound) of chlorin e6 (0.025 mg/mL) alone, the inhibitory effect on cell proliferation was significantly enhanced by the sonodynamic effect of chlorin e6 combined with paclitaxel in SPCA-1 cells (P＜0.05).Conclusion:Results of the present study demonstrated that paclitaxel enhanced the sonodynamic effect of chlorin e6 on the proliferation of human lung adenocarcinoma cell SPCA-1. Paclitaxel may be a promising sensitizing agent for the sonodynamic treatment of non-small cell lung cancer.