ANTICANCER RESEARCH 36: 5731-5742 (2016)

The Effects of Histone Deacetylase Inhibitor and Calpain Inhibitor Combination Therapies on Ovarian Cancer Cells

KAROLINA LAPINSKA1,2, GENEVIEVE HOUSMAN3, SHANNON BYLER4, SARAH HEERBOTH5, AMBER WILLBANKS1, ANUJA OZA1 and SIBAJI SARKAR1,6

1Cancer Center, Department of Medicine, Boston University School of Medicine, Boston, MA, U.S.A.;

2Quinnipiac University School of Medicine, North Haven, CT, U.S.A.;

3School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, U.S.A.;

4Department of Pediatrics, Children’s Hospital/Harvard Medical School, Boston, MA, U.S.A.;

5Vanderbilt University School of Medicine, Nashville, TN, U.S.A.;

6Genome Science Institute, Boston University School of Medicine, Boston, MA, U.S.A.

 

Abstract.

Background: Ovarian cancer is difficult to treat due to absence of selective drugs and tendency of platinum drugs to promote resistance. Combination therapy using epigenetic drugs is predicted to be a beneficial alternative. Materials and Methods: This study investigated the effects of combination therapies using two structurally different histone deacetylase (HDAC) inhibitors (HDACi), sodium butyrate and suberanilohydroxamic acid (SAHA), with the calpain inhibitor calpeptin on two characteristically different ovarian cancer cell lines, CAOV-3 and SKOV-3. Results: Suboptimal doses of HDACi and calpeptin produced several effects. Growth inhibition was enhanced and the epigenetically silenced tumor suppressor genes ARHI, p21 and RARβ2 were re-expressed. Methylation of specific CpG residues in ARHI were reduced. Cell-cycle progression was inhibited and apoptosis, as well as autophagy, were induced.

The phosphorylation of ERK and Akt were differentially effected by these inhibitors. Conclusion: The re-expression of tumor suppressors may sensitize ovarian cancer cells, which then undergo apoptosis and autophagy for cell death.

KEYWORDS:

Ovarian cancer; apoptosis; autophagy; calpain; cell cycle; combination therapy; epigenetic drugs; histone deacetylase inhibitors; methylation

PMID: 27793894

DOI: 10.21873/anticanres.11156

 

Supplement

According to the hallmarks of cancer (1), there are several pathways that contribute to carcinogenesis. The traditional methods of therapy address individual parameters and are not very successful. Thus, researchers perceived that it would be better to confront more than one issue at the same time. This concept generated the idea of combination therapy, which is more useful in treating cancer. Though combination therapy is clearly more advantageous than individual therapy, a significant number of patients still relapse. Researchers reason that the reasons for this anomaly is a heterogeneous population of cancer cells, especially in tumors.

Heterogeneous populations of tumor cells usually contain multiple mutations. The current paradigm states that a few among the several mutations, called driver mutations, propagate cancer. The problem with this idea is that different heterogeneous populations inside the same tumor can have different types of mutations (2, 3). It is extremely difficult to rationalize which mutations are driver mutations among the hundreds, and how diverse mutations initiate tumors of similar origins. This raises a question about how the initiation of similar types of cancers could be so disorganized, when the embryogenesis, developmental process, and the function and maintenance of somatic cells are so well organized (2, 3, 4,5,6). We have hypothesized before that mutations accelerate the development of tumors from already pre-formed cancer progenitor cells, which are derived from predisposed cells by alteration of epigenetic mechanisms in terms of differential histone modifications and CpG DNA methylation taken together, which we called “Epigenertic Switch” (2, 3, 5, 6, 7, 8). Our idea is supported by the fact that most of the heterogeneous tumors are derived from a few cancer stem/progenitor cells (2, 3). This notion is also supported by the fact that cancer stem/progenitor cell formation is a slow process. Recent studies also showed that indeed epigenetic alterations are involved in cancer stem/progenitor cell survival and progression (9, 10). The inherited mutations, such as BRCA1 and BRCA2 implicated in breast and ovarian cancers, do not necessarily produce breast and ovarian cancers in all persons who possess the mutations; however if they do succumb to the cancer, not every one develops it at the same age. In addition, recent studies have shown that conventional therapies including chemotherapy do not kill cancer stem cells and can help generate drug resistant cancer cells (7, 11, 12,13). Even after successful treatment, an apparent remission of cancer usually results in relapse. We believe that this relapse is caused by the presence of the cancer progenitor cells, which are not killed by conventional therapy, as well as by the presence of drug resistant cancer cells. In addition, we rationalize that as the conventional therapy does not address the issue of epigenetic mechanism alterations, the formation of cancer progenitor cells continues. We may imagine that after remission, even if some of the cancer stem cells still exist within the system, it would take some time for them to develop a full fledged cancer. This is why relapse of cancer takes a while, from months to years. There are some cases, which support this idea. For example, some of the acute myeloid leukemia’s (AML) are caused by mutation in DNMT3a and other genes (14,15). After treatment and complete remission, the cancer relapses. It is interesting to note that the higher level of methylation observed in AML patient cells was not reduced to the level of normal cells (14, 15), after treatment and remission, which suggests that formation of cancer progenitor cells does not stop even after therapy and remission due to higher methylation levels.

The combination therapy we have shown in this paper (16) is similar to what we observed before against both hormone responsive and triple negative breast cancer cells (16, 17). As there is no specific target in triple negative breast cancer, usually nonspecific and comparatively toxic platinum drugs are the choice for chemotherapy. In ovarian cancer the situation is similar. Usually, nonspecific drugs are the only choice. These types of drugs develop resistance. A recent study has shown that treatment with epigenetic drugs rendered platinum drug resistant ovarian cancer cells sensitive to the same drug (18). It has been observed by researchers that an epigenetic pathway regulates sensitivity of breast cancer cells to HER2 inhibition via FOXO/c-Myc pathways (19). Recent studies have also shown that treatment with epigenetic drugs is capable of killing breast cancer stem cells, which are usually resistant to conventional therapy (20). These similarities between breast and ovarian cancers led us to perform a comparative study of the genetic and epigenetic aspects of both cancers. We determined that breast and ovarian cancer of epithelial origin possess similar genetic and epigenetic alterations (5). That led us to postulate that breast and ovarian cancer progenitor cell formation could be produced by similar epigenetic mechanisms. Though not established yet, this analysis provides an opportunity to study the possible role of epigenetic alterations as a general mechanism of breast and ovarian cancer progenitor cell formation. Though we believe that individual types of cancer progenitor cells may harbor selective epigenetic alterations, those alterations should be similar if not identical to develop cancer/tumor of similar origin. This will explain why and how heterogeneous tumor populations with diverse mutations could be derived from a few cancer stem/progenitor cells.

In short, our current study (16) and other studies taken together, provides tremendous potential to determine how exactly cancer progenitor cells are formed. Elucidation of this mechanism provides a converging attitude towards management and cure of cancer, which we believe is a more organized phenomenon. This raises a possibility that inclusion of epigenetic drugs in combination with other type of conventional therapies may reduce the chance of cancer relapse, which is extremely difficult to achieve.

 

 

Karolina Lapinska: 1st Author

Sibaji Sarkar: Corresponding Author

 

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