Development of nimesulide analogues as dual tubulin and HSP27 inhibitor for treating female cancers

Tubulin and heat shock protein 27 (HSP27) are up-regulated in cancer cells, and play a critical role in cell division, and proliferation. Therefore, they are targets for discovery of anticancer therapy. The objective of this study is to design, characterize, and biologically evaluate the nimesulide analogues to combat female cancer such as ovarian cancer, and breast cancer. Herein, the nimesulide analogues are designed to target both tubulin and HSP27 functions. Ovarian cancer (SKOV3) and breast cancer (SKBR3) cell lines were used as surrogate models to test the nimesulide analogs biological activities using MTT assay. In the present study, four nimesulide analogues were designed, synthesized and the chemical structures were with the biological evaluation were studied. The synthesized agents were characterized by H-NMR, C-NMR, the molecular weight was confirmed using GC-MS technique, and melting point. Besides, the agent L4 structure was confirmed using X-ray crystallographic analysis. The present data revealed that nimesulide analogs have potent anticancer activity against SKOV3and SKBR3 cell lines. The IC50 values for both SKOV3 and SKBR3 cell lines treated with the agents showed a potent cell growth inhibition range of 0.23-2.02 μM and 0.50-3.73 μM respectively. In conclusion, the designed nimesulide analogues can target both tubulins, and HSP27 concurrently, and they are promising agents as future chemotherapy female cancers.


Introduction
The microtubules control many vital functions in the cells such as cell division, cell movement cellular trafficking, and many other biological functions. The main structural component of microtubules is the protein tubulin. [1][2] Given the importance of tubulin, it is potentially targeted for the discovery of natural and synthetic agents in cancer treatment. [1][2] Tubulin is a highly dynamic structured protein and is involved in cell division, movement, intracellular trafficking, maintenance, and mitosis. Alpha and beta polypeptide subunits form tubulin the rigid cylindershaped protofilament. [1][2] Thirteen protofilaments form a straw-shaped filament known as microtubules, which is a major component of the cytoskeleton and forms the spindle structure that appears during cell division. [1][2] Microtubule dynamics rely on the α-β-tubulin dimer's hydrolysis that binds α to GTP (minus end) and β to GDP (plus end). This cycle is important for microtubule dynamic stability and cell division. [1][2] The rapid growth of cancer cells leads to their high dependence on tubulin polymerization/ depolymerization, which makes tubulin a highly attractive target for anticancer drug design. [1][2] Several studies reported that microtubules inhibitor as anticancer agents, paclitaxel is an example of a drug used clinically for cancer treatment. It binds to tubulin and inhibits the polymerization/depolymerization process and causes mitotic arrest and then cancer cell death. However, after a good response to the initial treatments, most of the approved drugs become less effective due to the development of drug resistance in patients. 2,3 Therefore, further studies are required to discover anticancer agents to inhibit microtubules polymerization with minimal side effects.
Additionally, heat shock protein 27 (HSP27) is a small protein with 27 kDa that facilitates the refolding of the damaged proteins. 4,5 HSP27 responds to heat shock and cellular stress conditions when face carcinogens and functions as an anti-apoptotic agent. [6][7][8][9][10][11][12][13][14][15][16] HSP27 is found to be up-regulated in many cancers including liver, bladder, 17 pancreases, ovarian, and breast. 13-18 4 The previous reported that targeting HSP27 using antisense oligonucleotide -427 decreases HSP27 expression. Also, antisense oligonucleotide -427 in combination with chloroquine has been reported to decrease prostate cancer xenograft; herein, inhibition of HSP27 is a promising target in cancer therapy. 17,[19][20][21] Although the current clinically used anticancer drugs showed great benefits in female cancer treatments, there is a requirement for the development of novel anticancer drugs with enhanced pharmacological activity and ideal therapeutic index 10,19 . In the previous reports, the scientists established many potent agents that targeted HSP27 function and showed cytotoxicity against ovarian cancer cell lines. 22 Based on these dual reliable protein biomarkers 15 tubulin and HSP27. 8,9 In the last years, new agents were developed with dual targeting both tubulin and HSP27 proteins for breast cancer treatment using chaperonin and tubulin polymerization inhibition assays. 23,24 Recently, HSP27-HER2 dual inhibitor cholesterol conjugate was studied in our study using western blotting, and the prepared inhibitor can inhibit ovarian cancer cell lines. 25 Cyclooxygenase-2 is frequently expressed in many types of cancers exerting a pleiotropic and multifaceted role promotion of carcinogenesis and cancer cell resistance. 26 Nimesulide is considered a selective cyclooxygenase-2 inhibitor. In the last years, nimesulide was reported as an antitumor agent, besides its roles as an anti-inflammatory, pain killer, and antipyretic action. [27][28] Thus, numerous works were achieved on the nimesulide skeleton to obtain selective cyclooxygenase-2 inhibitors with potential antitumor action. [27][28] Further studies are required to address this assumption. Therefore, the current work aimed to develop novel, and potential compounds based on nimesulide structure figure 1 with targeting the ability to tubulin and HSP27 concurrently. The cytotoxicity of the design anticancer agent against both ovarian and breast cancer cell lines was investigated as a surrogate model for female cancers. The structure-activity

Results and discussion
The main structural component of microtubules is the protein tubulin. Tubulin is a potential target for the discovery of natural and synthetic agents in cancer treatment. 29 Moreover, HSP27 is found to be up-regulated in many cancers, and the inhibition of HSP27 was reported as a promising target in cancer therapy. [13][14][15][16][17][18][19][20][21] In the last year, nimesulide was reported as an antitumor agent [27][28] , further studies are required to address this issue. Therefore, in the present study, four nimesulide analogues were designed, synthesized and their chemical structures were elucidated and studied. The synthesized agents were characterized by 1 H-NMR, 13 C-NMR, the molecular weight was confirmed using GC-MS technique, and melting point. Also, agent L4 structure was confirmed using X-ray crystallographic analysis. Furthermore, the dual tubulin and HSP27 inhibition were investigated, besides the cytotoxicity against both ovarian and breast cancer cell lines.

Chemistry
According to the synthetic route shown in scheme 1, four nimesulide analogs were designed, synthesized and the chemical structures were shown in Table 1 with the biological evaluation results. The synthesized agents were characterized by 1 H-NMR, 13 C-NMR, the molecular weight was confirmed using GC-MS technique, and melting point. Besides, the agent L4 structure was confirmed using X-ray crystallographic analysis as shown in Figure 4.

Anticancer activities evaluation of agents targeting dual proteins tubulin and HSP27 in women cancer cell lines using MTT assay
Dual protein inhibitors were developed and evaluated for their potency in a panel of SKOV3 and SKBR3 cell lines using MTT assay and the IC50 values were calculated and summarized in Table   1. The cells were seeded at a concentration of 25 mg/L, and most of the agents showed desired anticancer activity. The data revealed that SKOV3 cell line adapted cell growth inhibition ranged from 0.20 µM to 2.02 µM. While SKBR3 cell line showed cell growth inhibition ranges from 0.50 µM to 3.70 µM. In sum, the data analysis of the preliminary bioassay indicated that the synthetic agents had potent anti-cancer activity and targeted dual proteins in both cell lines.

Targeting dual proteins in SKOV3 and SKBR3 cell lines using western blot assay
Western blot analysis was performed, and total cell lysates for SKOV3 and SKBR3 cell lines were extracted and treated with DMSO and separated by SDS−PAGE then transferred into nitrocellulose membrane. The membrane was blocked for one hour with NFDM, followed by being probed with the primary and secondary antibodies targeting HSP27 protein and tubulin respectively for one hour each. B-actin was used as our housekeeping control as depicted in Figure   3. The data revealed that two cell lines expressing HSP27 protein and tubulin. The results of the present study are in agreement with several previous studies reported that nimesulide analogs have anti-cancer properties. 27,28 Particularly, cyclooxygenase-2 is frequently expressed in many types of cancers, and promote the carcinogenesis and resistance of cancer cells to therapy. 26 However, the link between cyclooxygenase-2 inhibition and anticancer activity of nimesulide and its analogues is not clearly established and not fully understood. 27,28 In general, nimesulide and their analogues revealed both anticancer cyclooxygenase-2 dependent and independent mechanisms. 27,28 In the present study, dual tubulin, and HSP 27 inhibition by the developed nimesulide analogues might responsible for the cytotoxic effect against SKOV3 and SKBR3 cell lines. In this context, it has been reported that the disruption of microtubules functions induces cell cycle arrest and cell death. 29 As well, a previous study documented that, the dual inhibition of HSP27-HER2 can inhibit ovarian cancer cell lines. 25 Further studies are required for the development of new nimesulide analogues with improved anticancer potency. In addition, the confirmation of the anticancer effects of developed nimesulide analogues required additional in vivo studies including tumor-bearing female animals.

Materials and methods
Nimesulide analogs were synthesized in our laboratory. All chemicals and solvents used for the synthesis of compounds were commercially available and were used directly without the need for any preparation. All chemical spectral analysis was done at Kuwait University Research Sector Project Unit -RSPU. TLC was performed using Polygram SIL G/UV 254 TLC plates and visualized under ultraviolet light at 254-and 350nm, with hexane/ethyl acetate solvent. Column chromatography was performed using silica gel 60A of mesh size 40-60 µm. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were obtained using NMR spectrophotometer Bruker, DPX 600 at 600 MHz and 150 MHz in DMSO and in CDCL3 respectively. Mass spectra were acquired using a GC-MS DFS -Thermo spectrometer. IR spectra were obtained with a Jasco 6300 FTIR.
Determination of the melting point spectra done using a differential scanning calorimeter-Netzsch DSC 204 F1 Phoenix. The X-ray structure for Agent L4 was refined using the Bruker SHELXTL software package.

General procedure for the synthesis of compound L 2-5
2-Amino-5-nitrophenol (1 eq.) was used as our starting material and was mixed with K2CO3 (1 eq.) and 2,5-dichlorobenzyl chloride (1 eq.) in DMF to obtain compound A. In the next step, both sodium hydride (5 eq.) and methane sulfonyl chloride (1 eq.) was added to compound A (1 eq.) in dry DMF at room temperature and the reaction mixture was left overnight to obtain compound B.
This was followed by a methylation step in presence of methyl iodide and sodium hydride and DMF to produce compound C. Then, it was reduced in the presence of Zn (10 eq.), FeCl3 (4 eq.), DMF, and water to reduce the nitro group in compound C to an amine group and obtain compound D. Then, different moieties such as (1) 4-methoxybenzamide (2) 3,4-dimethoxybenzamide (3) 4iodobenzamide (4) naphthylamide were added with (1 eq.), K2CO3 (1 eq.), and 1:4 dioxane were added to compound D (1 eq.) to obtain the final compounds L2-5, which are our dual proteins inhibitors. The synthesis steps were carried out at room temperature. Water was used to quench all the reactions' steps. Reaction steps were tested by TLC to check the end-point. Compounds were purified by column chromatography.
L4 agent structure was confirmed using X-ray crystallography as depicted in Figure 4 and Table   2.    Table 3 Bond lengths and angles (Å) for agent 3 (C22H19Cl2IN2O4S).

MTT assay
MTT assay was performed by monitoring the reduction of yellow MTT reagent to a blue product.
Briefly, after culturing the cells in the monolayer cell culture plates, cells were harvested and then seeded with RPMI1640 medium into 96-well plate and incubated overnight. Then, cells were treated with different concentrations of anticancer agents in four replicates each and incubated for 72 h. DMSO control wells received concentrations equal to those in the drug-treated cells. 100µL of 0.5mg/mL of MTT reagent in fresh medium was added after the removal of the old medium.
Then, cells were incubated in the CO2 incubator at 37°C for one hr. Supernatants were removed from the wells, and then 100µL DMSO were added on the reduced MTT dye. The final absorbency measurement was determined using a plate reader at 570 nm and the values were normalized to controls.

Statistical analysis
The cytotoxicity of the agents was determined using Graph Pad Prism software (Graph Pad Software Incorporated) and Microsoft Excel (Microsoft Corporation) was used to determine the statistical information. IC50 values were normalized using nonlinear regression analysis software.

Western blot assay
Confluent women cancer cells' dishes (SKOV3 and SKBR3) were washed with PBS and harvested by the scraper. The supernatants were then removed after centrifugation and the collected cell pellets were lysed by mixing them with RIPA, PI, and EDTA 150µL. The cells were then vortexed and centrifuged for 60 minutes. Cell lysates were normalized using poly-acrylamide gel electrophoresis in the presence of 0.1% SDS. The gel contains the proteins that were transferred to the membrane followed by blocking with NFDM and PBST. The membrane was washed 3-4 times with PBST for 5 minutes. Then, the membrane was incubated with the primary antibody (target-HSP27, tubulin) for 24 hours. The next day the membrane was washed 3-4 times with PBST and incubated with the secondary antibody (horse radish peroxidase) for one hour. The membrane was again washed with PBST 3-4 times to be prepared for the visualization of the bands by chemiluminescence, and it was striped using B-actin.

Conclusion
In the present study, nimesulide analogues as dual tubulin and HSP27 inhibitor as anticancer agents were synthesized, and their structures are established by the chemical techniques. The developed nimesulide analogues can inhibit the function of tubulin and HSP27. Interestingly, MTT assay data revealed that both female cancer cell lines (SKOV3 and SKBR3) adapted cell growth inhibition.
The present data indicated that nimesulide analogues have cytotoxic activity at micro molar concentration and promising as future chemotherapy drugs. The future goal is to synthesis new agents and improves their biological activities to reach the nano-molar level.