| Cancer is being the first threaten of human healthy that seriously impact people’s lives. As the most common women cancer, breast cancer has brought painful experience to many women and families. The data of about 500,000 women dead from breast cancer in the worldwide, warned us the perniciousness of this cancer. The statistic data of China indicate that the new increased breast cancer cases occupied the top 2 of all new cancer cases. And the data will go on rising with the progress of diagnostic technology.In the last half century, the discoveries, developments and improvements of chemo-therapy drugs, have brought hope to thousands of patients and families. These progresses changed many patients living and extended their lives, and had being the good assistant of clinicians. But the side effects of chemo-therapy drugs, such as disgust, vomit, low immunity, cardiotoxicity, genetoxicity etc, also afflicted the patients, which limited its clinical application. Hundreds of scientists and clinicians tried hard to solve these questions, but so far, the reslut is still not satisfactory, and it is still the challenge of patients and clinicians.Pu-er tea, which is abundant in Yunnan, not only has long history of drinking, its health care functions have also been found and utilized. Recently its roles in cancer prevention attracted attentions of many researchers and clinicians. The researches of our laboratory, have proved that Pu-er tea could reduce the expression of mutant p53, inhibit the growth of p53 mutant mouse tumor cell lines, and then inhibit the cancer progression.But the studies on Pu-er tea combined chemo-therapy drugs treat cancer cells and normal cells, were limited. So in this study we used the breast cancer cell line T47D, MDA-MB-468, normal breast epithelial cell MCF-10A, colon cancer cell HT-29 as study objects to study the combination effects of Pu-er tea with doxorubicin, cisplatin, cyclophosphamide, paclitaxel on these cells. Firstly, the influence of the cell proliferation with combination dealing with by Pu-er tea and chemo-therapy drugs was checked by sulforhodamine B colorimetric assay (SRB assay). The results indicate that Pu-er tea combined with doxorubicin do not affect its harming functions to T47D, MDA-MB-468 and HT-29 cells, but could impair its toxicity to MCF-10A cell, the normal control cell line. Meanwhile, we observe the differences of MCF-10A cell growth between treated by doxorubicin alone and combination treated with Pu-er by microscope, and found that in combination dealing case, the survival cell number of MCF-10A was much more than doxorubicin alone dealing case. It also shows that combination treatment reduced the doxorubicin harming to normal cell line. However, there is no obvious protection when Pu-er tea combination with cisplatin, cyclophosphamide or paclitaxel.We also study the mechanism of this protection phenomenon in molecular levels. The results of western blot show that, after combination treatment the cleaved apoptosis signal proteins caspase8, caspase3 and PARP reduced remarkably, comparing to doxorubicin alone dealing case. It suggests that this protection may contributed by reducing the cell apoptosis arising by doxorubicin.At the same time, we paid our attentions to the changes of DNA damage signals. As we had expected, the signs of DNA damage, such as γ-H2AX, p53, phosphorylated-p53 (p-p53), reduced remarkably. These results indicate that after combination treatment the DNA damage reduces, which may also contribute to the protection. We also use pulse field gel electrophoresis (PFGE) assay to test the DNA damage, the results are consistent to western blot.Take consideration that Pu-er tea may interact with doxorubicin, which could affect doxorubicin getting into cells or impair its harm effect to normal cells, we design fluorescence assay to evaluate its possibility. The outcome of the fluorescence shows that Pu-er tea has no affection of the doxorubicin getting into cells process. We try to confirm the interactions between the two substances through high performance liquid chromatography (HPLC) assay, but the drug concentrations we using is lower than the HPLC test limitation, so whether there is interaction has still not confirmed.In addition, we studied the roles of Pu-er tea to the T47D and HT-29 cell lines, which have different mutant p53 expression, and found no obvious affection on p53 expression when treated by Pu-er. We also explored the ingredients of Pu-er tea: quercetin, EGCQ caffeine and gallic acid, combined with doxorubicin treat MCF-10A cells, observe no protection phenomenon, which means that there may exist other component of Pu-er contribute to the protection.In conclusion, combination treatment of Pu-er tea and doxorubicin reduced the toxicity of doxorubicin to normal cells, and almost had no effect to the killing of cancer cell. This protection was contributed by reducing cell apoptosis and DNA damage, but not coming from preventing doxorubicin getting into cells. When Pu-er tea combined with other chemo-therapy drugs, there were no remarkable functions. These studies provide solid basic for the following Pu-er tea combined with doxorubicin on animal assays. |
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