Activation of AMP-Activated Protein Kinase by 3,39-Diindolylmethane (DIM) Is Associated with Human Prostate Cancer Cell Death \u3cem\u3eIn Vitro\u3c/em\u3e and \u3cem\u3eIn Vivo\u3c/em\u3e

There is a large body of scientific evidence suggesting that 3,39-Diindolylmethane (DIM), a compound derived from the digestion of indole-3-carbinol, which is abundant in cruciferous vegetables, harbors anti-tumor activity in vitro and in vivo. Accumulating evidence suggests that AMP-activated protein kinase (AMPK) plays an essential role in cellular energy homeostasis and tumor development and that targeting AMPK may be a promising therapeutic option for cancer treatment in the clinic. We previously reported that a formulated DIM (BR-DIM; hereafter referred as B-DIM) with higher bioavailability was able to induce apoptosis and inhibit cell growth, angiogenesis, and invasion of prostate cancer cells. However, the precise molecular mechanism(s) for the anti-cancer effects of B-DIM have not been fully elucidated. In the present study, we investigated whether AMP-activated protein kinase (AMPK) is a molecular target of B-DIM in human prostate cancer cells. Our results showed, for the first time, that B-DIM could activate the AMPK signaling pathway, associated with suppression of the mammalian target of rapamycin (mTOR), down-regulation of androgen receptor (AR) expression, and induction of apoptosis in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. B-DIM also activates AMPK and down-regulates AR in androgen-independent C4-2B prostate tumor xenografts in SCID mice. These results suggest that B-DIM could be used as a potential anti-cancer agent in the clinic for prevention and/or treatment of prostate cancer regardless of androgen responsiveness, although functional AR may be required

B-DIM activates AMPK signaling and inhibits AR expression in prostate tumor xenograft tissues.

<p>ICR SCID mice were implanted with C4-2B cells and treated with 5 mg/mouse of B-DIM by oral gavages daily for 4 wks. Tumor tissues were analyzed by immunohistochemistry using anti-phosphor-AMPKα (T172), phosphor-ACC (S79) or AR antibodies. The stained sections were visualized under the microscope (400×amplification). Unlike solvent-treated control, mice treated with B-DIM presented with activation of AMPK and significant loss of AR in tumor sections.</p

AMPK inhibitor Compound C can block AMPK activation by B-DIM in prostate cancer cells.

<p>Prostate cancer C4-2B and LNCaP cells were pre-treated with 20 µM of AMPK inhibitor Compound C (CC) for 6 hours, followed by co-treatment with indicated concentrations of B-DIM for 3 hours. Cell extracts of the treated cells were immunoblotted for anti-phosphor-AMPKα, phosphor-ACC or β-actin antibodies.</p

Treatment with B-DIM or metformin inhibits prostasphere-forming ability.

<p>C4-2B cells were treated with indicated doses of B-DIM (A, B) for 6 days. Treatment with different concentrations of metformin served as controls (C, D). After 6 days, prostasphere numbers were counted under the microscope and the proportion of sphere-generating cells was calculated by dividing the number of cells seeded by the number of prostaspheres (A, C). Prostaspheres from C4-2B cells treated with B-DIM (B) or metformin (D) were photographed and the results showed that 10 and 25 µM of B-DIM significantly reduced size and numbers of prostaspheres. n = 6, * P<0.05, ** P<0.01.</p

B-DIM activates the AMPK pathway, resulting in inhibition of AR and PSA expression and induction of apoptosis in prostate cancer cells.

<p>Human prostate cancer C4-2B (A) or LNCaP (B) cells were treated with indicated concentrations of B-DIM for 3 hours to measure protein levels of phosphor-AMPKα, AMPKα, phosphor-Raptor, phosphor-ACC, phosphor-mTOR, or for 24 hours to measure protein levels of AR, PSA or PARP cleavage by Western blot analysis. Measurement of β-actin served as loading controls. The numbers underneath the Western results of phosphor-AMPKα indicate quantified normalized phosphor-AMPKα and β-actin ratios.</p

B-DIM enhances the effects of AR inhibitor casodex in prostate cancer cells, associated with increased activation of AMPK.

<p>Prostate cancer C4-2B and LNCaP cells were treated with 100 µM of casodex, 40 µM of B-DIM alone or a combination of casodex and B-DIM for 24 hours, followed by Western blot analysis using anti-phosphor-AMPKα, AR, PARP or β-actin antibodies.</p