Tests in vitro and in vivo suggest blocking PKCδ may in addition boost efficacy of chemotherapy.

Scientists claim that blocking a protein known as PKCδ protects the kidneys from cisplatin-related nephrotoxicity  and may in addition act to boost the chemotherapeutic effects of the anticancer drug. In vitro and in vivo studies showed that PKCδ plays a critical role in the mechanism by which cisplatin causes kidney damage.

The work was conducted by Zheng Dong, Ph.D., at the Medical College of Georgia and Charlie Norwood VA Medical Center, and colleagues. Findings are reported in The Journal of Clinical Investigation in a paper titled “Inhibition of PKCδ reduces cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in mouse models of cancer.”

The biochemical pathways responsible for renal tubular cell injury and death are not yet understood. It is not known whether the same pathways that drive cisplatin’s chemotherapeutic effects also cause the toxic side effects and whether it might be possible to block the drug’s damaging effects without impacting on its tumor-killing properties.

PKCδ is expressed in a wide variety of cells and tissues and has been implicated in the regulation of cellular processes ranging from signal transduction to apoptosis. To investigate the potential effects of the protein in cisplatin-related pathways, Dr. Zong’s team started by analyzing PKCδ activity in a mouse model in which a single dose of cisplatin induces acute kidney injury and renal failure.

They found that in these animals cisplatin administration led to a 2–3-fold increase in PKCδ kinase activity. This activity was triggered after just one day, preceded signs of kidney injury, and continued during the development of severe kidney damage and renal failure.

Interestingly, the increase in kinase activity wasn’t due to higher PKCδ expression; levels of total PKCδ remained largely constant. Rather, the researchers detected PKCδ phosphorylation at tyr-311, which started at day one of cisplatin treatment. Studies in cultured renal proximal tubule cells (RPTCs) also showed that cisplatin induced rapid PKCδ phosphorylation and activation as well as translocation of PKCδ to the nucleus.

To identify the protein kinase or kinases responsible for PKCδ phosphorylation and activation during cisplatin nephrotoxicity, the team screened pharmacological inhibitors of various tyrosine kinases. Two inhibitors of the Src family tyrosine kinases, PP1 and PP2, suppressed cisplatin-induced PKCδ (tyr-311) phosphorylation, while a control compound PP3 had no effect.

Further co-immunoprecipitation studies showed that in control RPTCs, Src interacts with PKCδ and that this interaction is enhanced during cisplatin treatment both in renal cells and also in mouse kidneys during cisplatin nephrotoxicity.  The two inhibitors PP1 and PP2 partially blocked the interaction between PKCδ and Src in cell lines.

Adding a further piece to the mechanistic puzzle, the researchers showed that MAPKs including JNK, ERK, and p38 were activated in kidney tissues from cisplatin-treated wild-type mice. Conversely, in the kidneys of PKCδ knockout animals ciaplatin-induced JNK activation was markedly delayed, p38 activation was completely abolished, and ERK activation was transiently suppressed.

The increase in RPTC apoptosis following cisplatin treatment was also significantly reduced on administration of rottlerin, a pharmacological inhibitor of PKCδ  inhibitor. Conversely, blocking the other PKC isoforms PKCα, PKCβ, and PKCγ had no effect on cistplatin nephrotoxicity, the researchers found.

Importantly, the protective effects of rottlerin in cultured RPTCs were supported in experimental mice. Animals treated using both cisplatin and rottlerin demonstrated reduced renal tissue damage and apoptosis, in comparison with those treated using just cisplatin. Experimental PKCδ-knockout mice treated with cisplatin also suffered less kidney damage and demonstrated better renal histology than wild-type mice.

Because a role for p53 has previously been demonstrated in cisplatin nephrotoxicity and PKCδ has separately recently been implicated in p53 phosphorylation and activation, the authors hypothesized that PKCδ would phosphorylate p53, leading to p53 activation and consequent renal apoptosis. However, this was surprisingly found not to be the case.

Intead, cisplatin induced p53 phosphorylation downstream of PKCδ activation in both wild-type RPTCs  and in those isolated from PKCδ knockout mice. In addition, PKCδ (tyr-311) phosphorylation occurred independently to p53 in kidney tissues.

Functionally, cisplatin-induced apoptosis in PKCδ-knockout cells could further be suppressed by pifithrin-α, a pharmacological inhibitor of p53, whereas the PKCδ inhibitor rottlerin was not effective. Conversely, rottlerin but not pifithrin-α could suppress cisplatin-induced apoptosis in p53-knockout cells.  “Thus PKCδ and p53 appeared to be independently regulated during cisplatin nephrotoxicity,” the authors write.

The scientists do stress that PKCδ- and p53-mediated signaling pathways are not the only uniquely responsible pathways for cisplatin nephrotoxicity because 20%–30% of cisplatin-related apoptosis remained, even under the conditions of inhibition of both PKCδ and p53.

To try and find out whether inhibiting PKCδ would have benefits during cisplatin therapy for cancer, the researchers carried out tests in nude mice implanted with human ovarian cancer cells.  After allowing the cancers to start growing, the xenograft-bearing animals were then treated with either  cisplatin alone, cisplatin plus rottlerin, or saline (the control group).

After four weeks the two drug-treated groups both showed tumor shrinkage. While the cisplatin-only animals also demonstrated significant increases in markers of kidney damage, those treated with both cisplatin and rottlerin exhibited better renal function and lower levels of kidney tissue damage and apoptosis. Of the 11 cisplatin-treated animals, 10 died within 34 days, whereas treatment-related death in the cisplatin-rottlerin group took significantly longer, and three animals survived the entire observation period.

Similar tests carried out in immune-competent mice bearing mouse ovarian cancers were even more promising, suggesting that the innate immune system plays a role in helping to prevent drug-related damage . In these animals, adding rottlerin to cisplatin therapy similarly preserved kidney function, and improved survival to a greater degree than had been observed in the nude mice. 

“While all of the cisplatin-only-treated mice died within four weeks, approximately 40% of the animals of the cisplatin-plus-rottlerin group survived,” the researchers report.  Moreover, all the deaths were due to the cisplatin therapy and not due to tumor growth, as the untreated cancer-bearing control animals survived the observation period, even though their tumors were still growing.

The researchers found equivalent results when they added rottlerin or another, even more specific  PKCδ-inhibitor known as δV1-1 to cisplatin treatment in animals bearing testicular cancer grafts or human breast cancer tumors. In these animals the additional PKCδ inhibitor also helped reduce other chemotherapy-induced side effects including weight loss, suggesting that inhibiting PKCδ may have chemoprotective effects in other organs.

“We have identified PKCδ as a critical regulator of cisplatin nephrotoxicity, which can be effectively targeted for renoprotection during chemotherapy,” the authors conclude. “ We showed that early during cisplatin nephrotoxicity, Src interacted with, phosphorylated, and activated PKCδ in mouse kidney lysates.

“After activation, PKCδ regulated MAPKs but not p53 to induce renal cell apoptosis. Thus, inhibition of PKCδ pharmacologically or genetically attenuated kidney cell apoptosis and tissue damage, preserving renal function during cisplatin treatment.”

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