Assessment of Sanglifehrin-based Cyclophilin Inhibitors for Hepatocellular Carcinoma Treatment

Abstract

Among all cancer types, liver cancer has the second highest mortality rate. HCC is the most common liver cancer and patients usually have a very poor prognosis. The standard-of-care drug Sorafenib extends the life expectancy with just two to three months, making the development of new drugs for the treatment of HCC an important area of research.

Here, the pharmaceutical candidates SfA1 and SfA2 were examined as potential drugs for treatment of hepatocellular carcinoma (HCC). SfA1 and SfA2 are bona fide cyclophilin inhibitors, hypothesized to inhibit proliferation and survival of cancer cells.

The drug candidates were evaluated by analyzing the drugs’ effect on cell cycle phases, glycolysis, clonogenic capacity and protein expression. Cell cycle analysis was carried out through the flow cytometric BrdU-technique. The drugs were found to inhibit the cell cycle at the G1/S and G2/M checkpoints. Glycolysis stress tests on drug-treated cells resulted indicative data on the drugs’ lack of involvement in glycolysis. Clonogenic assays, in which the cells were continuously treated with the drugs, showed that the drugs decrease clonogenicity in a dose-response dependent manner. SfA2 was found to be more potent than SfA1. Additionally, differences were found in cell line sensitivity, where the most sensitive cell line has a wild type p53 gene. Western blot analysis targeting CypA, CypB, IMPDH1 and IMPDH2 showed a decreasing protein content of CypB and IMPDH2 with increasing drug doses, suggesting these proteins are important in the drugs’ mechanism of action.

This work provides a foundation for further studies on the mechanism behind the anticancer properties of SfA1 and SfA2. Of particular interest is CypB and IMPDH2 involvement, for which supportive evidence was found through this work. The drugs’ effects on several cell properties suggest the involvement of multiple pathways.
Among all cancer types, liver cancer has the second highest mortality rate. HCC is the most common liver cancer and patients usually have a very poor prognosis. The standard-of-care drug Sorafenib extends the life expectancy with just two to three months, making the development of new drugs for the treatment of HCC an important area of research.

Here, the pharmaceutical candidates SfA1 and SfA2 were examined as potential drugs for treatment of hepatocellular carcinoma (HCC). SfA1 and SfA2 are bona fide cyclophilin inhibitors, hypothesized to inhibit proliferation and survival of cancer cells.

The drug candidates were evaluated by analyzing the drugs’ effect on cell cycle phases, glycolysis, clonogenic capacity and protein expression. Cell cycle analysis was carried out through the flow cytometric BrdU-technique. The drugs were found to inhibit the cell cycle at the G1/S and G2/M checkpoints. Glycolysis stress tests on drug-treated cells resulted indicative data on the drugs’ lack of involvement in glycolysis. Clonogenic assays, in which the cells were continuously treated with the drugs, showed that the drugs decrease clonogenicity in a dose-response dependent manner. SfA2 was found to be more potent than SfA1. Additionally, differences were found in cell line sensitivity, where the most sensitive cell line has a wild type p53 gene. Western blot analysis targeting CypA, CypB, IMPDH1 and IMPDH2 showed a decreasing protein content of CypB and IMPDH2 with increasing drug doses, suggesting these proteins are important in the drugs’ mechanism of action.

This work provides a foundation for further studies on the mechanism behind the anticancer properties of SfA1 and SfA2. Of particular interest is CypB and IMPDH2 involvement, for which supportive evidence was found through this work. The drugs’ effects on several cell properties suggest the involvement of multiple pathways.

Popular Abstract

In this project, two new drug candidates; SfA1 and SfA2, were assessed as potential treatment for liver cancer. The drugs were effective in slowing down the growth of cancer cells by blocking the cell cycle. Additionally, the drugs were found to decrease the expression of the proteins CypB and IMPDH2, involved in DNA synthesis, indicating that these proteins are involved in the drugs’ mechanism of action.

The aim of this project was to evaluate a new type of drug for the liver cancer hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer and accounts for 800 000 deaths/year. The current prognosis for HCC patients is poor and only early stage cancers patients can really benefit from the therapeutic treatments. In later stages, the drug Sorafenib is currently used. As this drug only extends the life expectancy with 2-3 months, new drugs are needed to provide more effective treatment for the patients.

The drug candidates used in these experiments inhibit a type of proteins called cyclophilins. Cyclophilins have many roles in eukaryotic cells, one of which is to assist in protein folding by controlling and accelerating it. In normal cells, this is usually not vital as the spontaneous folding of proteins is fast enough. However, in cancer cells, cyclophilins play an important role. Cancer cells divide very rapidly, which means that they need to produce correctly folded proteins much faster than normal cells. In addition to this, cyclophilins have been found to be more abundant in the liver than in most tissues. With this background, the hypothesis of this project was that inhibition of cyclophilins would be effective as HCC treatment.

The work was planned around two main theories about the mechanism of action of the drugs. The first one was that the drugs effect the cells’ metabolism through glycolysis, as previous research has reported on the involvement of cyclophilin D (CypD) in cell respiration, and inhibitory effects of the known cyclophilin inhibitors Sanglifehrin A and Cyclosporin A on CypD. The second hypothesis was that the drugs inhibit a protein called IMPDH2, which is involved in the synthesis of guanine, a DNA building block. Through this, IMPDH2 inhibition was hypothesized to generate an inability to synthesize DNA, resulting in a block in the cell cycle.

The first hypothesis was tested through an acute and a chronic glycolysis stress test. In these, the drugs’ effect on glycolysis was assessed by measuring the glycolysis rate under different cellular conditions in drug-treated cells. The chronic test showed no difference in glycolysis among the cells treated with different drug doses. Yet, it resulted in a lower glycolysis in drug treated cells than in control cells. This, however, is likely due to the slowed cell proliferation, which, consequently, slows down glycolysis as the cells require less energy. The acute test enabled elimination of this factor. The data from the acute test showed a very low absolute difference between control and treated cells, with no clear correlation to drug dose. In summary, the glycolysis stress test indicated that glycolysis is not a main pathway through which the drugs act. Additionally, the data suggests that CypD is not the main target protein.

To assess the second hypothesis, a cell cycle analysis was carried out. Previous data indicated that the drugs cause cell cycle arrest, meaning the drug treated cells are prevented from passing through the cell cycle in a normal manner. To find which phase of the cell cycle the drug candidates affect, a cell cycle analysis was carried out on cells treated with SfA1, using the BrdU method and flow cytometry. SfA1 was found to prevent the cells from going through the cell cycle at certain checkpoints, called the G1/S and G2/M checkpoints. Instead of passing through the cell cycle in a normal manner, the cells accumulated in the G1 and G2 phases. A possible explanation for this could be that the drug inhibits the synthesis of nucleotides used as building blocks for DNA, and could thus be conclusive with the hypothesis on IMPDH2 inhibition.

Clonogenic assays were used to assess the drugs’ effect on the cells’ ability to grow and form colonies. In these assays, HCC cells were continuously treated with different doses of SfA1 or SfA2. The drug candidates were shown to prevent HCC cells from forming colonies in a dose-response dependent manner, meaning that the higher the drug dose, the lower the number of colonies was present at the end of the experiment. This indicates that the drugs can prevent HCC cells from dividing. SfA2 was found to be a more potent drug than SfA1. Differences were found in cell line sensitivity, where the cell line with a wild type p53 gene was found to be the most sensitive. P53 is known to be involved in cell cycle regulation and tumor suppression, and could therefore be an important factor for the mechanism of action of SfA1 and SfA2.

The Western blot technique was used to look at the protein expression of CypA, CypB, IMPDH1 and IMPDH2 in cells treated with SfA1 or SfA2. The protein expression of CypA appeared to be unaffected, which indicated that changes in CypA expression is not involved in the mechanism of action. The protein expression of IMPDH1 was found to slightly decrease after SfA2 treatment. The expressions of CypB and IMPDH2 were found to clearly decrease with increasing SfA1 and SfA2 doses. This suggests that these proteins, CypB and IMPDH2 in particular, may be involved in the drugs’ mechanism of action.

In conclusion, the drugs were found to inhibit cell growth and to cause cell cycle arrest. Supportive evidence was found for the involvement of CypB and IMPDH2 in the drugs’ actions. Glycolysis stress experiments ruled out the hypothesis on glycolysis inhibition as a mode of action of SfA1 and SfA2.

The data produced during this project is a small contribution towards the investigative work required to map the mechanisms of action of SfA1 and SfA2. Support presented for the involvement of several proteins indicates that the drugs might work through multiple pathways. This is a promising property in the search for non-resistance inducing drugs, as the cancer cells will need extensive adaptations to override the drugs’ actions.

To confirm the early indications of possible important pathways presented here, and to consider other possibilities, further mechanistic studies are needed. An additional cell cycle analysis is planned where an external supply of DNA building blocks is hypothesized to restore the normal cell cycle properties. Further Western blot studies, including looking at the p53 gene product expression, are interesting possibilities to confirm the data presented here and to evaluate the importance of the p53 gene status in cell line sensitivity. Additionally, a tissue microarray study to look at the expression of the protein of interest in human tumor samples is planned.

• author: Johannesson, Mikaela
• supervisor: Eskil Elmer; Magnus Hansson; Michele Tavecchio
• organization: Applied Microbiology; Biotechnology
• course: KMB820 20171
• year: 2017
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: applied microbiology, teknisk mikrobiologi
• language: English
• id: 8909718
• date added to LUP: 2017-05-31 15:58:28
• date last changed: 2017-05-31 15:58:28