| Objectives Cancer cells differ from normal cells in that they can immortally grow by escaping apoptosis. However, most of cancer cells acquire apoptosis-resistance ability by inactivating up-stream signals that trigger caspases cascades not by directly removing caspases, suggesting that caspases might be vital to carcinogenesis. We developed biosensors that can measure in real time caspases’ activity in live cells and applied them in breast cancer cells to screen basally activated caspases. We indentified a low level of activated caspase-3/7 that was indispensable to the growth of breast cancer cells, supported by our further results obtained with sh RNA and chemical compounds against caspase-3/7. Caspase-3/7 is the principal executioner in the apoptotic pathways, and they usually undergo cascade amplification.Main Methods Frist, we constructed plasmids,then the above plasmids were amplified, prepared lentivirus and constructed stabe cell lines. We cultured cells and divided the cells into experimental and control groups. By cell count and soft AGAR cloning experiment testing Caspase- 7 expression level of MCF- 7 cell growth, proliferation and clone formation ability.We detected the expression levels of Caspase-7and other proteins by Western Blot. Using Caspase-7 inhibitor Z-DEVD-fmk MCF-7 cell, the cell count and soft AGAR cloning formation experiment, observation of inhibitors on MCF- 7 cell growth, proliferation and clone formation ability. Apoptosis induced by TNFα. Sugar-free DMEM culture culture cell, observe the MCF- 7 / vector, the MCF- 7 / caspase- 8 dn of sugar-free stimulates the development of the ability to survive. AT101 stimulate cells, observe the Caspase- 8, 7, and the relationship between the mitochondrial membrane permeability.Results 1. The breast tumor cells exist in the limited Caspase activation.2. Caspase- 3/7 of the growth of breast cancer cells is a must. Inhibition of the activity of Caspase- 3/7 can inhibit MCF- 7 cell anchorage- dependent and anchorage- independent of cell growth. 3. Caspase- 3/7 help to boost the breast tumor cells on the surrounding metabolic micro environment adaptability. 4. Caspase- 3/7 in breast cancer cells plays a very important role in energy generation, the inhibition of the activity of Caspase- 3/7 inhibited MCF- 7 cell ATP production. 5. Controllable activated Caspase- 3/7 May be activated by Caspase- 8, but trace activation of Caspase- 7 does not lead to cell apoptosis. Expression of Caspase- 8 dn in small MCF- 7 cell spontaneous apoptosis of cells but can suppress the apoptosis induced by TNFα. 6. Caspase-8 rate can improve the energy metabolism of breast tumor cells. 7. Low doses of AT101 can promote MCF-7/Caspase-8DN a large number of apoptosis.Conclusions Our research shows that exist in the MCF- 7 in breast tumor cells Caspase- 7 bits of activation, the activation is controllable, and necessary to cell growth proliferation, soft AGAR experiments showed Caspase- 7 of MCF- 7 colony-forming ability is also necessary, prompt Caspase- 3/7 in breast tumor metastasis and likely degradation plays an important role in the process. Caspase- 3/7 in breast tumor cells also play an important regulating role in glucose metabolism, under the condition of high low glucose and lactic acid, inhibition of the activity of Caspase- 3/7 can quickly kill the MCF- 7 mammary gland cells, prompt Caspase- 3/7 help to boost the breast tumor cell metabolism of the surrounding environment adaptability. Another inhibition of Caspase- 3/7 of the activation can inhibit the production of ATP, breast tumor cells prompted Caspase- 3/7 in breast cancer cells plays a very important role in energy metabolism. Limited activated Caspase- 3/7 May be activated by Caspase- 8, Caspase- 8 May effect on mitochondrial membrane, the membrane permeability increased, but not widely MOMP formation, activation of asmall amount of Caspase- 7, limited activated Caspase- 7 not lead to cell apoptosis, but the breast tumor cell survival and metastasis and coping with adverse metabolic microenvironment plays an important role. |
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