The Reversion Effect On Esophageal Cancer Cell Malignancy By Liposomal Quercetin

Quercetin, 3,3',4',5,7- pentahydroxyflavone, belongs to the flavanoid and is widely distributed in the plants. The biological effects of quercetin has been investigated worldwide. The known effects include anti-oxidant, anti-inflammation, anti-bacteria/virus and inhibiting neoplasm growth and metastasis.The quercetin is water insoluble, but could be dissolved in DMSO reagent. The toxic effects were reported when lower than 0.05% of DMSO solution was applied in cell culture. However, the safety of quercetin solution in DMSO is unknown. To increase the solubility and improve biological activity, liposomal quercetin was used in vivo studies..The esophageal squamous epithelial carcinoma (ESEC), is developed from malignant squamous epithelial tissue. One of the highest incidences of ESEC is in China. The cancer cells are characterized with resistance to hypoxia, chemical drug and radiation. The tumor cells have strong proliferation capacity, high expression of cyclin D1 inhibition of PTEN suppressor gene expression, which in turn, inhibits the PI3k/Akt signal transduction pathway. As the result, cell proliferation is inhibited.. Over expressions of c-met and VEGF in human esophageal cancer cells could be induced under hypoxia condition. It implies that modulating oxidative stress (ROS) could be an effective method to inhibit the tumor cell proliferation. It was reported that ROS was involved in the hepatoma apoptosis induced by quercetin. Moreover, quercetin plays dual biological functions. For normal cells, it shows anti-oxidant effect, while for tumor cells, it enhances the oxidative stress. Therefore, quercetin has theroputic and preventive effects on cancer.The present study was designed to investigate the reversion effect of malignancy induced by liposomal quercetin in the Eca109/9706 cells and explore the potential mechanism.Aims:1. To compare the biological effects of the liposomal quercetin prepared by different ways.2. To explore the malignancy reverse effect and its potential mechanism in the Eca109/9706 cells induced by liposomal quercetin mediated with oxidative stress.Methods:1. The liposomal quercetin was prepared mainly by evolutionary evaporation technique. The liposomal quercetin, (LQ₁) was prepared by using chloroform and methanol as the solvent, the liposomal quercetin. The second liposomal quercetin (LQ₂) was prepared by using chloroform and DMSO as the solvent. The liposomal quercetin stained with oil-red was identified under microscope. The non-liposomal quercetin (nLQ) was prepared by using DMSO as the solvent.2. The suppressing rate of cultured Eca109/9706 cells was detected by MTT assay. Eca-109/9706 cells were divided into four groups: LQ₁, LQ₂, nLQ and negative control groups (C).3. Eca109/9706 cells were incubated with liposomal quercetin in cell culture media. After 48-hour-incubation, NO activity was measured by using histochemistry assay and free radical level was tested by using corresponding kit.4. After 48-hour-exposure to liposomal quercetin, the expression and cellular localization of PTEN, c-met, VEGF and cyclin D1 in Eca109/9706 cells were examined by using immunohistochemistry assay.5. After the exposure, the Eca109/9706 cell protein was extracted by using Trizol reagent and separated by running SDS-PAGE. 6. Western blotting was carried out to identify and semi-quantify c-met, VEGF, cyclin D1, and PTEN.β-actin was used as the internal control.7. The statistical analysis was accomplished with SPSS12.0 software., the data, x|-±s, were analyzed with ANOVA in difference between groups and correlation analysis with Bivariate for correlated indexes was detected, the significant different level wasα=0.05.Results:1. The solubility and homogeneity of LQ₂ was better than LQ₁. Cheking under the microscope by using oil lens, the LQ₂ particle appeared as central quercetin micro-particle surrounded by red-stained lipids.2. The suppressing rate of Eca-109/Eca-9706 cells was in the order of LQ₂ group> LQ₁ group>nLQ group>control group (P<0.05).3. The NO activity intensity was in the order of LQ₂ group>LQ₁ group >nLQ group >control group (P<0.05).4. The cellular free radical level was in the order of LQ₂ group>LQ₁ group>nLQ group>control group (P<0.05). It was also noticed that the NO activity was positively correlated with free radical level (r₁₀₉=0.956 , P<0.05, r₉₇₀₆=0.957, P<0.05).5. The protein expression of c-met, VEGF, PTEN and cyclin D1 was found in Eca109/9705 cell cytoplasma as well as in nuclei by using immunohistochemistry assay. The Immuno-reaction intensity of c-met, VEGF, and cyclin D1 was in the order of control group> nLQ group>LQ₁ group> LQ₂ group (P<0.05). On the contrary, the PTEN-IR intensity was in the order of LQ₂ group>LQ1 group>nLQ group>control group (P<0.05).6. The western blotting showed that the molecular sizes of c-met, cyclin D1, VEGF, andβ-actin were 145 kDa , 36 kDa, 21 kDa and 42 kDa respectively. The intensity of immunoblotting signals for c-met-IR, VEGF-IR and cyclin D1-IR was in the order of control group>nLQ group>LQ₁ group>LQ₂ group (P<0.05). The intensity of PTEN-IR was in the order of LQ₂ group>LQ₁ group>nLQ group> control group (P<0.05), which was in the reversed order. The results of immunohistochemistry and western blotting were positively correlated in the four groups (r=0.89-0.95, P<0.05).Conclusions:1. The biological effect of the liposomal quercetin two (LQ₂) is predominant over that of the liposomal quercetin one (LQ₁).2. LQ₂ has stronger the suppressing effect on Eca109/9706 cells than LQ₁ does.3. Exposing Eca109/9706 cells to the liposomal quercetin for 48 hours, NO activity and the free radical level were increased.4. The liposomal quercetin mediated with oxidative stress contributes to the reverse of the high expression of c-met and VEGF, which has been found to be enhanced under hypoxia condition.5. The malignancy-reverse effect induced by the liposomal quercetin on Eca109/9706 cells acts mainly through up-regulating the expression of the tumor suppressor, PTEN, which can inhibit the PI3k/Akt signal transduction pathway, and down-regulate expression of cyclin D1.