| Prostate cancer is a kind of ordinary tumor often afflicts old men. Traditional treatments always transform the prostate cancer into hormone refractory carcinoma which restricts conventional treatment. Like normal prostatic epithelium, growth and survival of early prostate carcinoma depends upon androgen stimulation. Androgen-ablative therapy, achieved by surgical or medical castration, induces programmed cell death (apoptosis) of androgen-dependent tumor cells . However, not all prostate tumor cells are affected by androgen ablation and in time a significant portion of patients treated with hormonal therapy relapse with androgen-independent prostate carcinoma. Now there has not any effective therapies for androgen-independent prostate carcinoma. Menadione (VK3) is considered a natural vitamin K, but rather a synthetic analogue that acts as a provitamin. The anticancer activity of menadione has also been demonstrated in a number of in vitro studies using both rodent, and human cancer cell lines. Historically the effectiveness of menadione against cancer was believed to be due to oxidative stress via redox-cycling of the quinone to produce reactive oxygen species(ROS) such as the hydroxyl radical, superoxide radical, and hydrogen peroxide. Supporting this theory is the observation that menadione increased oxidative stress in malignant cells. NF-κB is one of the most important nuclear transcription factor, it is a key position between the oxidative stress and apoptosis. NF-κB is active in the nucleus and is inhibited through its sequestration in the cytoplasm by the inhibitor of κB(IκB). NF-κB becomes active once it is released from IκB. Free NF-κB for translocation to the nucleus and activation of its target genetic programs. So we study on the growth inhibition by VK3 in androgen-independent human prostate cancer cell lines PC-3M and research the possibly mechanisms, from this experiment we want to provide a new therapy for androgen-independent human prostate cancer. OBJECTIVE: To explore and study the mechanisms that inhibit growth and apoptosis induced by VK3 in PC-3M cells. MATERIALS AND METHODS:1.Cell culture;2.Detect the inhibitory rate of cells growth by MTT assay; 3.Observe the cells change by inverted microscope; 4.Detect apoptosis rate by AO assay; 5.Using flow cytometric PI-staining to detect apoptosis in PC-3M cells. 6. DCFH-DA fluorescence spectrophotometry was used for ROS detection. 7.Detect the GSH-Px and CAT mRNA expression were assayed using RT-PCR; 8.Detect the antioxidatation through antioxidant NAC against VK3; 9.Location the activation of NF-κB P50/P65 by immunocytochemical stain ; 10.Detect the content changing of NF-κB P50/P65 in cytoplasma by Western Blotting. Result: 1. Cell growth was inhibited after the addition of VK3 and the inhibitory effect was dependent on time and dosage. VK3(60μM) cansignificantly inhibited PC-3M cells (P<0.01). 2. After the addition of VK3, we observed the changes of cells in the inverted microscope. Compare with the control cells, after addition of VK3 cells cytoplasma recovery, cell rounding, cells division were obviously decreased . Apoptosis cells which are shrink and floating and characterized by apoptosis bodies are observed. The changes of cells shape were dependent on time and dosage. 3. We observe the changes of cells under the confocal microscopy with VK3 treatment after AO staining . We can observe marked changes of apoptosis include nuclear condense, nuclear side collection, caryolysis, nuclear fragmentation . Low dose of VK3 (≤60μM ) can induce PC-3M cells to apoptosis, and large dose(≥120μM)of VK3 can lead to necrosis. 4. It can be observed that G1/G0 arrest in cell cycle and hypodiploid by FCM after the treatment of VK3(60μM) for 12h. Inhibitory ratio of 60μM Vk3 was 27.44%. 5. NAC antioxidation assay , PC-3M cells were inhibited by mM level NAC, and NAC has not inhibition effect to PC-3M cells in μM level. 40μM dose of NAC can significant against inhibition by VK3 on PC-3M cells. 6. To examine the production of intracellular ROS, we monitored DCF fluorescence. We observed and increase in the level of DCF fluorescence after PC-3M was treated for 2-8h with VK3(60μM). ROS produced by VK3 can be significant decreased by 40μM dose of NAC(P<0.001). Antioxidant NAC inhibited VK3-stimulated ROS...
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