Metformin Augments Panobinostat's Anti-Bladder Cancer Activity by Activating AMP-Activated Protein Kinase

Abstract

Panobinostat, a histone deacetylase inhibitor, induces histone acetylation and acts against cancer but attenuates its anticancer activity by activating the mammalian target of rapamycin (mTOR) pathway. AMP-activated protein kinase (AMPK) is a cellular energy sensor that reportedly inhibits the mTOR pathway. The antidiabetic drug metformin is also a potent AMPK activator and we investigated whether it augmented panobinostat's antineoplastic activity in bladder cancer cells (UMUC3, J82, T24 and MBT-2). Metformin enhanced panobinostat-induced apoptosis and the combination inhibited the growth of bladder cancer cells cooperatively in vitro and in vivo . As expected, metformin increased the phosphorylation of AMPK and decreased the panobinostat-caused phosphorylation of S6 ribosomal protein, thus inhibiting the panobinostat-activated mTOR pathway. The AMPK activation was shown to play a pivotal role in the combination's action because the AMPK inhibitor compound C attenuated the combination's anticancer activity. Furthermore, the AMPK activation by metformin enhanced panobinostat-induced histone and non-histone acetylation. This acetylation was especially remarkable in the proteins in the detergent-insoluble fraction, which would be expected if the combination also induced endoplasmic reticulum stress.

Simvastatin-Romidepsin Combination Kills Bladder Cancer Cells Synergistically

“…In the present study, inhibition of ER stress by cycloheximide markedly impaired the combination's ability to cause histone acetylation and induce apoptosis, suggesting that the ER stress induction played a pivotal role in the combination's action. This ER stress-histone acetylation sequence is also consistent with our previous results that there is a crosstalk between histone acetylation and ER stress induction [29,30,73,74,80]. The decreased expression of HDACs is thought to be a consequence of the ER stress induction according to the previous studies [29,30,73,74,80].…”

“…Unexpectedly, the simvastatin-induced AMPK activation was further promoted by romidepsin, which might also play a role in enhancing the histone acetylation. This AMPK activation was thought to be due to ER stress induction by the combination because the ER stressor tunicamycin increased the expression of AMPK, which is consistent with the previous reports [29,30,73,74]. ER stress is caused by the accumulation and aggregation of unfolded proteins [75], and excessive ER stress causes apoptosis and kills cancer cells [76][77][78].…”

“…2h). Our previous studies showed that ER stress activates AMPK [29,30], so we thought that the combination of romidepsin and simvastatin would also induce ER stress and thereby enhance AMPK activation. As expected, the combination induced ER stress cooperatively (Fig.…”

“…AMPK is a molecule which controls cellular energy homeostasis and metabolism essential for cancer proliferation [65,66] and therefore its activation is considered to be a novel anticancer mechanism [2][3][4][5]. Several studies demonstrated that simvastatin inhibits tumor growth by activating AMPK [12][13][14], and AMPK activation has been shown to induce histone acetylation [27][28][29][30]. We therefore thought that combining simvastatin with an HDAC inhibitor would kill cancer cells effectively by inducing histone acetylation cooperatively.…”

“…We thought that simvastatin would activate AMPK and the simvastatin-romidepsin combination would kill bladder cancer cells effectively by inducing histone cooperatively. We also investigated the role of endoplasmic reticulum (ER) stress induction in the combination's anticancer activity because histone acetylation is closely related to ER stress induction [29,30]. Furthermore, simvastatin is known to activate peroxisome proliferator-activated receptor (PPAR) γ, a regulator of fatty acid storage and glucose metabolism [31][32][33], which plays a crucial role in bladder cancer proliferation [34][35][36].…”