Drug Repurposing of Pantoprazole for Its Anticancer Activity Focusing on Its Anti-Angiogenic Potential

Cancer is one of the leading disease which affects the current human population worldwide. It is caused when cells divide in an uncontrollable manner and it damages the body tissues^1,2. Even though there are many drugs available in the market, treatment resistance still pose a major threat in the field of oncotherapy. Drug repurposing is the novel strategy which uses an existing drug candidate for a new medical condition other than it was indicated for.  The side effect of a drug can also be utilized for its potential to treat a new disease. Since its safety and efficacy studies were already done for other medical condition, the risks and costs for conducting those studies can be reduced³. The entire drug development process can also be skipped. Based on the phenotypic benefits, drug repurposing can identify new compounds without drawing its mechanism of action⁴. These drugs directly reach the preclinical testing and it progress directly to Phase II clinical trials⁵. The risk of failure is minimum with repurposed drugs compared to drugs discovered via traditional approach. From the standard drug discovery process the pharmacokinetic, toxicology and safety data can be extracted. One of the example is Thalidomide, which was once used to treat morning sickness in pregnant women banned due to its terratogenecity is repurposed for the treatment of leprosy and multiple myeloma. In such cases the benefit-to–to-risk ratio has to be considered. Amphotericin B is an antifungal drug now used for leishmaniasis. Tamoxifen is an anticancer drug now used for leishmaniasis therapy. Astemizole is an antihistamine now repurposed for malaria therapy. Minoxidil is anti-hypertensive utilized for treating alopecia. Asprin, an NSAID now also indicated for myocardial infarction. There are many such drugs which are in clinical trials and also available as therapy which implies the significance of drug repurposing. Pantoprazole is a proton pump inhibitor given for the treatment of heartburn, acid reflex and gastro eosophageal reflex disorder. Several studies pointed out the anticancer activity of pantoprazole in in-vitro culture. Weidong Shen et.al., studied the effect of pantoprazole on human gastric adenocarcinoma SGC7901 cells through regulation of phospho?LRP6 expression in Wnt/?-catenin signaling and found out that pantoprazole has anti-proliferative and pro-apoptotic effects. Zhoulei li et al., studied the action of Pantoprazole and Vitamin C Targeting Tumor Microenvironment Conditions Improves Anticancer Effect in Metastatic Castration-Resistant Prostate Cancer. Zhen-ning L et.al., found out that Pantoprazole pretreatment elevates sensitivity to vincristine in drug-resistant oral epidermoid carcinoma in vitro and in vivo.

MATERIALS AND METHODS

I. Molecular Docking

Docking is done to find out the binding affinities between the candidate drug and the receptor. It is a preliminary screening tool. Here Pantoprazole is docked with the Vascular endothelial growth factor receptor to find out whether the molecule binds with the protein and thereby understanding whether the drug has got role in the angiogenesis process or not^6,7,8.

The software used is GLIDE developed by Schrodinger.

II. MTT Assay

It is the preliminary invitro anticancer test to assess the cytotoxic potential of drugs. Here breast cancer cell lines, Human cervical cancer cell lines are used for the study. Cyclophosphamide is employed as the standard^9,10.

1. HeLa cells were grown in the medium at 37?C in an incubator.

2. The cells were counted using a haemocytometer and the number of cells were adjusted to 20,000 cells per ml.

3. In a 96 well plate 200 µl of the cells were seeded in triplicate.

4. Pantoprazole were given in the concentrations of 10,20,30,40,50 µg/ml. The standard drug Cyclophosphamide were also given in these concentrations.

5. Cells are incubated for one day and then washed with PBS.

6. MTT solution was added to each well in the concentration of 20 µl, incubated for four hours. The colour change from yellow to purple is noted.

7. After replacing the solutions from each microtitre well, DMSO is added and the cells were kept at room temperature for half an hour.

8. The optical density was measured using a plate reader at570nm and then percentage viability of the cells was calculated.

III. Evaluation of response of sprouting seeds

The germination of mung beans, Vigna radiate (L.) (Fabaceae) is also a preliminary evalauation method for assessing the cytotoxic potential of the drug¹¹. When the seeds transition from a metabolically inactive quiescent state to the rapid cell division stage it imbibes water. The seeds then shift from G1 to G2 phase that procedes cell division and the growth of the radicle. The weight increase in the seeds is thus indicated as the measure of seedling growth. Hence, the evaluation of germination response of mung beans can be employed as the inexpensive method of screening cytotoxic drugs.

1. Mung beans of uniform weight were taken for the analysis. The weight of each seed is taken and recorded.

2. The seeds were placed in the microtitre plate well and different concentrations of test drug and standard drug solutions were added to it. Distilled water and DMSO control were also set up. It is then kept in an open space for 24 hours.

3. In the concentrations of 10,20,30,40,50 µg/ml, Pantoprazole was given. Cyclophosphamide was also given in the same concentration.

4. After 24 hours and 48 hours the weight of each seeds were noted and then calculate the percentage inhibition of sprouting.

Percentage inhibition = WtD – WtE/ WtD – WtMI

WtD – Wet weight of seed in distilled water

WtE – Wet weight of seed in experimental sample

WtMI – Wet weight of seed in maximum inhibition.

Angiogenesis is the formation of new blood vessels from the endothelial cell progenitors^12,13. Angiogenesis plays a major role in the tumor progression and it is one of the hall marks of cancer. Chorioallantoic membrane is a membrane composed of chorion and allantois and it supplies nutrients to the embryo during its developmental stage¹⁴. CAM assay is a good model to study angiogenesis and anti-angiogenesis properties of drugs^15,16. The degree of vascularization is studied in this test and compared.

1. 3 day embryonated eggs were collected from nearby poultry farm.

2. The eggs were gently wiped with 70% IPA

3. Air sac was located using a light souce and a cut was made using a forceps. The cut

should be of 1 cm in diameter.

4. Using a syringe carefully remove the albumin.

5. The eggs were covered with a plastic tape and keep in the incubator for 7 days

at 37?C.

6. At the 10th day Pantoprazole was added at the concentration 50?g/?L. The standard

drug was also added at the same concentration. The drug was added using a

micropipette onto a sterilized filter paper disc kept on the CAM.

7. The eggs were covered with plastic tapes and then kept in a incubator for 3

days.

8. At the 13th day eggs were taken from the incubator the shells were removed

and the contents were put in a petridish and photographs were taken.

1. Molecular Docking

Table 1: Percentage vaiability of HeLa cells after 24 hours of drug treatment

            
           
       
    Figure 4: Percentage vaiability of HeLa cells after 24 hours of drug treatment

III. Evaluation of response of sprouting seeds

The sprouting response of seeds after treatments with Pantoprazole and Cyclophosphamide were studied in the 24 hour and 48 hour time intervals. The percentage inhibition were calculated and compared with the help of graph. The data indicates that Pantoprazole has inhibitory effect on seed sprouting and the inhibition increases with increase in the dose of drug. Cyclophosphamide being an established cytotoxic drug has good inhibitory property than the test drug. But in higher concentrations, Pantoprazole also shows good percentage inhibition of sprouting response. It indicates the cytotoxic activity of Pantoprazole.

           
           
       
  Figure 5: Comparison of percentage inhibition of sprouting after 24 hours of drug treatment

Macroscopic observations of Pantoprazole treated egg, Cyclophosphamide treated eggs, untreated control and DMSO control were done. The untreated egg showed normal pattern of blood vessel formation with proper branching pattern. DMSO control has slight breaks in the blood vessels but no significant changes were seen. However, the treatment with 50?g/ml concentration of Pantoprazole produced a significantly visible inhibition in the formation of blood vessels. The branching pattern of blood vessel formation was very poor in the given concentration. The standard drug Cyclophosphamide also show a clear cut inhibition of angiogenesis.