Cytotoxic and apoptotic potential of some coumarin and 2-amino-3-carbonitrile selenophene derivatives in prostate cancer

3-acetyl coumarin derivatives (1a-d) are formed as a result of condensation of salicylaldehyde derivatives and ethyl acetoacetate and were converted into coumarin-selenophene hybrid compounds (2a-d) in the basic medium by modified Gewald reaction in the presence of malononitrile and selenium. Products are characterized by nuclear magnetic resonance (NMR). The prepared compounds are screened for their anticancer activity against DU-145 cell line. In addition, selected target compounds are evaluated for apoptosis and oxidative stress on DU-145 (prostate carcinoma) cell lines.


Introduction
Cancer is a disease that occurs in any part of the body and spreads rapidly. It ranks second among the diseases that cause human deaths in the world. About 9 million people die each year from cancer. The most common types of cancer are breast cancer and colorectal cancer in women and lung cancer and prostate cancer in men. Although many drugs have been developed by scientists to treat cancer, they cause the cells to show resistance besides their fatal side effects. For this reason, demand for new cancer drugs acting with different mechanisms is increasing day by day.
Selenophene compounds are five-membered heterocyclic aromatic compounds containing selenium element, which is very important and unique for the body. Proteins containing selenium are called 21st century protein (selenocysteine). It is known in the literature that compounds containing selenophene show biological activity such as anticancer [17][18][19], anti-HIV [20] and antifungal [21,22]. In addition, due to the high cytotoxic effect of compounds containing selenium, the new hybrid compounds that form with these compounds can be very effective and selective against cancer cells.
There are few studies on the synthesis of new compounds formed by combining coumarin and selenophene and investigating their biological activities. In 2017, Domracheva et al. synthesized the compounds of selenophenocoumarin and selenophenoquinolinone, examined in vitro cancer activities, and clarified the mechanism [23]. In 2020, Erşatır et al. synthesized 8 new coumarin-selenophene hybrid compounds and investigated the anticancer activities of these compounds and coumarins with starting compounds in the MCF-7 breast cancer cell line. They determined that the new compounds were more active than the starting materials [6]. By combining two or more pharmacofores with a single covalent bond and molecular architecture, we can minimize the side effects that may occur while increasing the efficiency and activity [24]. Most cancer medications such as Voreloxin, Cefatrizine, and Quarfloxin have also been created for this purpose. In the light of all this, the present study is to combine two effective and biologically active compounds with a single covalent bond to obtain compounds that will be effective against various cancers, particularly prostate cancer. The main purpose of this study is to examine the activities of coumarin compounds in different cancer lines and to test whether they show higher biological activity with the synergy created by combining hybrid compounds compared to the starting compounds. This study is a continuation of our previous study where we investigated the synthesis of eight coumarin-selenophene hybrid compounds and their antiproliferative activities on the MCF7 breast cancer cell line. Four of these compounds showed very good activity on this cell line. We examined the antiproliferative activities of these four compounds and their starting substances on the DU145 prostate cancer cell line. Also, Caspase 3, 8, and 9 activities of these four hybrid compounds and starting substances were measured, MDA (3,4-Methylene dioxy amphetamine), and glutathione levels were tested.

Synthesis of coumarin and coumarin-selenophene derivaties
All coumarin and coumarin-selenophene derivatives were synthesized based on our previous study (Table 1) [4]. Their characterization studies were carried out by nuclear magnetic resonance (NMR) spectra, which is specified in supporting information.

Antiproliferative activity
DU-145 human prostate cancer cell line was obtained from the American Type Culture Collection (ATCC, USA). Cells were routinely cultivated in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 U/cm 3 ), and streptomycin (100 mg/cm 3 ) at 37 °C and 5% CO 2 . Protein levels were measured by the Lowry method [25]. Cell line was selected based on many clinical trials showing the activity of selenium compounds in the reduction of prostate cancer [26].
The cells were seeded at a density of 3 × 10 4 cells per plate in 16 well E-Plate (xCeLLigence, ACEA Biosciences), then completed with medium and incubated overnight in a 5% CO 2 incubator at 37 °C. Subsequently, cells were treated with different tested (2a-2d, 1a-1d) at different concentration (0, 5, 10, 20, 40 µM). All experiments were performed in triplicate. Untreated cells were used as control groups. Doxorubicin (Sigma) was used as a reference drug.

Lipid peroxidation measurement
MDA level of cell lysates were determined according to Ohkawa et al. The principle of the method depends on the measurement of the pink color produced by the interaction of thiobarbituric acid with MDA as a result of lipid peroxidation. The color density was measured via spectrophotmether at 532 nm. The results were expressed as nmol/mg protein [27]. 2.5. Glutathione measurement GSH levels were measured using the method reported by Beutler et al. The reaction of glutathione and DTNB (Ellman's reagent), generated 2-nitro-5-mercapto-benzoic acid. The color density was measured via spectrophotometer at 412 nm. The results were showed as μmol/mg protein [28].

Results and discussion
Coumarin compounds belong to benzopyrone family of plant secondary metabolites and very important for their therapeutic potential in cancer treatments. Their derivatives have shown wide variety of biological activities such as antioxidant, antiproliferative, antimicrobial, antiinflammatory, and antituberculosis. Although chemotherapy has been shown to be the most appropriate option against to cancer, it has been known to have deleterious systemic side effects. In order to avoid these side effects, new drug candidate needs to be designed [29,30]. In this study, novel potential anticancer agents were synthesized and tested on prostate cancer which is second most common cancer among men worldwide. Then, caspase enzyme activities and antioxidant properties were determined.

Antiproliferative activity
The significance of coumarin-selenophene derivatives shows antiproliferative activity against prostate cancer [31]. The cytotoxicity of coumarin and coumarin-selenophene derivatives was determined by xCELLigence system. Cell proliferation was determined by differences in cell-impedance variations when treated tested compounds on DU-145 cells. The results of xCELLigence assay IC 50 values are summarized in Table 2 and Table 3. It appears that hybrid compounds that are active even from the reference substance used in the previous study are also active in the DU145 cell line. Hybrid compounds also appear to be active in the DU145 cell line from the self-forming starting compounds. While the hybrid compound without substituents in the phenyl ring is the most active compound, the activity decreases in the presence of electron withdrawing groups at the 6th carbon. Myers et al. reported that 5 days of coumarin treatment inhibits the proliferation of two malignant prostate cell lines (DU145 and LNCaP) [32]. 7-hydroxycoumarinyl gallate ester showed high antiproliferative activity, which was superior to gallic acid, against DU-145 cell line [33].

Apoptosis
To disclose the molecular mechanism that is involved with apoptosis inducement, we measured Caspase 3, 8, and 9 levels in DU-145 cells treated with various concentration of coumarin (1a-1d) and coumarin selenophene derivatives (2a-2d) by ELISA method. The expression of Caspase 8 is involved in extrinsic pathway of apoptosis. Furthermore, the activation of Caspase 9 is a characteristic feature of intrinsic apoptosis pathways. Caspase 3 and 9 expression levels increase after treatment with 20.0 mM of 2a was statistically significant (Figure 1). Caspase 8 level also increased but it was not significant. Other tested compounds did not exhibit significant effect on Caspase 3, 8, and 9. According to the data were obtained 2a induced on the extrinsic pathway of apoptosis (Figure 1). Umar et al. have reported that 4flourophenylacetamide-acetyl coumarin induced apoptotic cell death by ROS-evoked p53-mediated caspase-dependent pathway in A549 cells [34].

Biochemical analysis
Ersatir et al. showed that coumarin-selenophene derivatives have antiproliferative activity against to MCF-7 breast cancer line [4]. Increased oxidative stress is related to weakening of the cellular defence mechanism. Cancer cells generate reactive oxygen species (ROS) more than healthy cells. Several drugs used in cancer therapy kill cancer cells by ROS production, and today many researchers are working on developing natural or synthesized cancer therapeutic agents that  increase ROS production. High concentration of reactive radicals causes lipid peroxidation, protein modification, and DNA damage causing apoptosis or necrosis. Malondialdehyde level is the most popular and reliable markers of oxidative stress and the antioxidant status in cancerous patients. In this study, 2a significantly increased MDA levels in a dose-dependent manner (Figure 2). Hacioglu et al. proved that ZnSO 4 caused a concentration-dependent increase in oxidative stress, apoptosis [35].  Kar et al. demonstrated that there is a correlation between increased oxidative stress and decreased cell growth and induction of apoptosis [36].
Kim DH et al. reported that ROS generation leads to apoptosis in human colon cancer HCT116 cells [37]. GSH has a tripeptide structure, which is an important antioxidant in cytosol.
GSH has many cellular functions such as antioxidant defence via direct interaction with ROS or via activities of detoxication enzymes like GSH peroxidases and GSH-S-transferases.
GSH depletion trigger to initiating an oxidative stress. Intracellular GSH levels are very important for cell death mechanism. In this study, we showed correlation between descending concentration of 2a compound and GSH levels ( Figure 2). Interestingly, GSH can regulate Caspase 3 and 9 catalytic activity as well as their proteolytic activation [38].

I. Instrumentation Details
Unless noted otherwise, all of compounds were used as provided without further purification. All of compounds were obtained from Merck and Sigma-Aldrich. Multiplicities are indicated, s (singlet), d (doublet), t (triplet), q (quartet), sept (septet), m (multiplet), br s (broad singlet). Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gel-precoated aluminium sheets (Type 60, F254, Merck, Darmstadt, Germany) using hexane/ethyl acetate 80-20 (4:1, v/v) and the spots were detected by exposure to UV lamp at λ254 nanometer for few seconds. The chemical names given for the prepared compounds are according to the IUPAC system.