Berberine Radiosensitizes Human Esophageal Squamous Cell Carcinomas By Downregulating RAD51 Expression

[Background]Esophageal squamous cell carcinomas (ESCC), which have a high incidence in East Asian countries, usually have poor prognosis. As for treatment of many other types of cancer, chemotherapy and radiation therapy are often applied to patients with ESCC. Radiation therapy can significantly relieve the symptoms and improve the quality of life in patients. But there are many limitations in radiation therapy, such as tumor recurrence after radiotherapy, radiation resistance, and side effects. Therefore, enhancing radiosensitivity of tumors and reducing radiation side effect is one of the major medical challenges.Berberine, an isoquinoline alkaloid component in several Chinese herbs including Huanglian, has been shown to have antimicrobial, anti-inflammatory, anti-diabetic and anti-angiogenesis and cholesterol-lowering effects. A large number of studies also showed that berberine possesses anti-tumor effect in many kinds of cancer, such as cervical cancer, esophageal cancer, oral cancer, colonic cancer, prostate cancer, leukemia, melanoma, osteosarcoma and glioblastoma. The underlying mechanisms include inhibition of protein synthesis, induction of cell cycle arrest, and apoptosis. Recent studies have shown that berberine could directly inhibit IKK activity in tumor cells, inhibit the phosphorylation and transcription of P65 and suppress the constitutive NF-kB activation. In addition to its direct tumor-killing effect, berberine was also reported to have radiosensitizing effect on lung cancer cells by inducing autophagy. In addition, berberine may suppress tumor growth by inhibiting angiogenesis.[Objective]The purpose of this study is to determine whether berberine has radiation sensitizing effect on esophageal squamous cell carcinomas, and to characterize the mechanisms involved.[Methods]1,MTT assay was used to determine the proliferation of esophageal cancer cells and human normal fibroblast(HNF) cells.2,Clonogenic assay was employed to assess the survival and proliferation potential of cells treated with berberine and/or ionizing radiation.3,The level of DNA double-strand breaks(DSBs) and the capacity of homologous recombination were determined byγ-H2AX foci and RAD51 foci, respectively by immunofluorescence.4,Western blotting and real-time PCR were used to determine the expression of RAD51,Ku70 and Ku86.5,Flow cytometry was used to analyze cell cycle distribution of berberine treated-cells6,Luciferase reporter assay was used to evaluate whether berberine regulates promoter activity of RAD51.7,RNA interference was used to verify that RAD51 downregulation radiosensitizes esophageal cancer cells. 8,The expression of RAD51 in human primary esophageal squamous cell carcinoma tissues and normal tissues surrounding the carcinoma was assessed by immunohistochemical stainings.[Results]1,Berberine inhibits the growth of KYSE30,KYSE450 and HNF cells in a time-and dose-dependent manner. But HNF cells have a higher tolerance than KYSE30 and KYSE450 cells. At low concentrations, berberine had minimal toxicity.2,Berberine pretreatment radiosensitizes KYSE30 and KYSE450 cells. In contrast, berberine had no radiosensitizing effect on HNF cells.3,Berberine pretreatment prolongs the persistence of ionizing radiation-induced DNA DSBs. Low concentration(15μM) of berberine treatment alone was not found to induce obvious DNA damage in terms ofγ-H2AX foci induction.4,The level of RAD51 protein, but not those of Ku70 and Ku86, was remarkably decreased in KYSE30 and KYSE450 cells that were treated with berberine.5,Berberine treatment did not significantly alter the distribution of cell cycle. Therefore, the reduced expression of RAD51 after berberine treatment is not due to changes in cell cycle distribution.6,Ionizing radiation induced an up-regulation of RAD51 in ESCC cells. Upregulation of RAD51 by IR can be attenuated by berberine.7,The level of RAD51 mRNA was downregulated.Luciferase reporter experiments showed that the promoter activity of RAD51 was significantly reduced in KYSE30 and KYSE450 cells that were treated with berberine when compared to control.8,RAD51-downregulation by RNAi radiosensitizes esophageal cancer cells. Therefore, the radiosensitizing effect of berberine on esophageal cancer cells was primarily mediated by the downregulation of RAD51.9,RAD51 is commonly overexpressed in human primary ESCC specimens while having no detectable or low expression in normal tissues surrounding the carcinoma.[Conclusions]Berberine at low concentrations substantially radiosensitized esophageal squamous cell carcinoma (ESCC) cells. It acts by downregulating RAD51, a key player in homologous recombination repair, leading to impairment in the repair of DNA double-strand breaks (DSBs).Our findings thus uncovered a hitherto unrecognized biological activity of a commonly used and inexpensive drug and indicated that berberine, with its relatively low toxicity, may be used as an adjuvant in cancer radiation therapy.