Anti Cancer Potential of some Indole based Quinoxaline Derivatives against Dalton’s Lymphoma (DL) cells

 

Shivam Bajpai¹*, Sundaram Singh²

¹Department of Chemistry, Bipin Bihari College, Affiliated To Bundelkhand University,

Jhansi- 284001, U.P., India.

²Department of Chemistry, Indian Institute of Technology, Banaras Hindu University,

Varanasi – 221 005, U.P., India.

 

ABSTRACT:

Indole based quinoxaline derivatives (2a-f) were synthesized and the therapeutic potential of the synthesized compounds (2a-f) was investigated against Dalton’s Lymphoma (DL) cells.  It was observed that treatment of DL cells with quinoxaline derivatives (2a-f) showed significant decrease in cell viability, altered morphology, nuclear disintegration and DNA fragmentation into low molecular weight DNA fragments and thus characteristic of apoptosis. In this view, the synthesized compounds may be used as potential therapeutic regimen to treat this type of cancer.

 

 

 

INTRODUCTION: 

The indole nucleus is a well-known N- containing heterocyclic ring system widely present in naturally occurring alkaloid-type products and synthetic molecules of interesting bioactivities.¹ While, quinoxaline^2,3 is versatile nitrogen containing heterocyclic moiety that posses a variety of biological activities, Its core structure was found in several naturally occurring compounds such as flavor-enzymes, riboflavin, molybdopterines and antibiotics of streptomyces.⁴ The diversity in the biological response of quninoxaline derivatives has gained more attention of many scientists and researchers to explore this skeleton to its multiple potential against several diseases and pathological conditions.⁵ Substituted quinoxalines are an important class of benzoheterocycles, that constitute the building blocks of wide range of biologically active compounds, possess anti-cancer^6-8, anti-malarial⁹, anti-bacterial^10,11, anti-fungal¹², anti-tubercular¹³, anti-leishmanial¹⁴, antidepressant¹⁵, potent anti-thrombotic¹⁶, anti-analgesic and anti-inflammatory activities.^17,18

 

 

On the view of the above, we investigated herein anti-tumor/anti-cancer^19-25 potential of some quinoxaline derivatives (2a-f) on Dalton’s lymphoma cells.

 

MATERIALS AND METHODS:

Materials:

All chemicals were procured from Aldrich, USA and E. Merck, Germany and used without further purification. RPMI 1640 culture medium was purchased from HiMedia, Mumbai, India and Fetal Bovine Serum (FBS) was obtained from Invitrogen, Grand Island, NY, USA. MTT [3-(4, 5-dimethylthiazol 2-yl) -2, 5-diphenyltetrazolium bromide], Con-A (concanavalin-A), proteinase-K and chelerythrine were purchased from Sigma chemical company, Bangalore, India. DNA ladder were purchased from Promega, Masison WI, USA. All other chemicals stated otherwise were obtained either from HiMedia, Mumbai, India or Super Religare Laboratory (SRL), Mumbai, India.

 

Cell culture and dose optimization:

Dalton’s lymphoma cells (DL cells) were cultured in RPMI-1640 supplemented with 10% FBS at 37⁰C in 5% CO₂. Growth of microbes was blocked using penicillin (100-100 µg/ml), streptomycin (100-100 U/ml), L-glutamine (4mM), 1% nonessential amino acids, and 1% of sodium pyruvate. For dose optimization, DL cells were treated with the compounds (2a-f) and incubated for 48 hr to test cell viability and followed by time kinetics. Cells were also cryopreserved for future reference.

 

MTT-assay:

The effect of the synthesized compounds (2a-f) on cell viability was assessed. To assess cell viability, cells were seeded on 96 wells plate at a density 4.0×10⁴ cells/well using RPMI-1640 enriched with Fetal Calf Serum (10% v/v). Cells were incubated for 48 hr with increasing concentration of compounds (2a-f) i.e. 10-400 µg/ml and chelerythrine (10 µg/ml) was used as a standard. Cells incubated with medium only were used as negative control. Cell viability was marked by the conversion of tetrazolium salt MTT to a coloured formazan by the action of mitochondrial dehydrogenase enzyme. Optical density of colour product was read at 595 nm using a Bio-Rad spectrophotometer. Optical density of untreated cells was used as negative control reference. The viability was determined using the following formula.

                                               _{Mean OD}

_{Percent Cell Viability = --------------------------- x 100}

                                             _{Control OD}

 

Assessment of cytotoxicity:

To assess the toxic effect of the compounds (2a-f) on DL cells, cells (1×10⁶ cell/ml) were treated with 200 µg/ml of compounds (2a-c, 2e and 2f) 100 µg/ml of compounds (2d) for 12 hr, 18 hr and 24 hr respectively. Thereafter cells and trypan blue dye (1:1 ratio) was mixed and observed under inverted light microscope. The percent of dead cells were determined using following formula.

                                  _{Number of Dead Cells}

_{Dead Cells (%) = -------------------------------- x 100}

                                   _{Total Number of Cells}

 

Determination of apoptosis:

Morphological assessment:

(a) Giemsa-Eosin staining:

To study the morphology of DL cells, cells were treated with the compounds (2a-f) for 12 hr, 18 hr and 24 hr in 5% CO₂ atmosphere. Cells were washed gently and uniformly smeared on neat and clean slides thereafter fixation and permealization in 4% formaldehyde solution were followed. After fixation, cells were washed properly and stained with Geimsa (which stain nucleic acid) and a counter stain eosin (cytoplasm staining dye). Further, cells were observed under simple light microscope to studied membrane blebbing, bulging and nuclear changes.

 

 

(b) EtBr- Acridine staining:

To assess morphology, cells were treated with 200 µg/ml of the compound (2a, 2e and 2f) and 100 µg/ml of the compounds (2b, 2c and 2d) for 12 hr, 18 hr and 24 hr in 5% CO₂ atmosphere. Thereafter cells were washed, smeared uniformly on neat and clean slides, permealized in 4% formaldehyde solution freshly prepared in PBS (0.01M). After fixation, cells were stained with EtBr (DNA binding dye) and a counter stain acridine orange (cytoplasm staining dye). Live/apoptotic cells were examined under Fluorescence microscope (Zeiss, India, Pvt. Ltd., Bangalore, India).

 

(c) Hoechst-33342 staining:

To study nuclear morphology, cells were incubated with 200 µg/ml of compounds (2a, 2e and 2f) and 100 µg/ml of the compounds (2b, 2c and 2d) for 12 hr, 18 hr and 24 hr in 5% CO₂ atmosphere. Treated cells were washed gently, smeared uniformly on neat and clean slides, fixed and permealized in 4% formaldehyde solution and thereafter stained with Hoechst-33342 (10µl for 1 million cells). Thereafter, cells were observed under fluorescent microscope at ̴ 460nm band pass filter to study nuclear changes (Zeiss, India, Pvt. Ltd., Bangalore, India).

 

Assessment of DNA fragmentation:

To assess DNA fragmentation, cells were treated with 200 µg/ml of the compound (2a, 2e and 2f) and 100 µg/ml of the compounds (2b, 2c and 2d) for 12 hr, 18 hr and 24 hr in 5% CO₂ atmosphere, washed and lysed in 1000 µl of lysis buffer [10mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% Nonidet P-40 (NP-40), and 100µg/ml proteinase-K and 100 µg/ml RNase] for overnight at 55 ⁰C in water bath. Apoptotic DNA was isolated and resuspended in Tris- EDTA buffer. Estimated concentration of DNA (2-5 µg/well) was separated electrophoretically on 2% agarose gel containing EtBr (0.5µg/ml). Microphotographs were taken under gel documentation system (Bio-Rad, India, Pvt. Ltd.).

 

Statistical Analysis:

For statistical calculation data were taken as mean ±SE. One-way ANOVA followed by Bonferroni as post-hoc test was used for statistical calculations. Data were considered to be significant at p< 0.05 as applicable.

 

RESULT:

Chemistry:

The quinoxaline derivatives (2a-f) were synthesized²⁶ and chemical structures of respective synthesized compounds were confirmed by their spectral data (IR, ¹H-NMR, ¹³C- NMR and elemental analysis).²⁶

 

Scheme 1: Library of synthesized compounds which were investigated under DL cell

 

Effect of the compounds (2a-f) on the cells viability:

In order to evaluate the effect of compounds (2a-f) on DL cells viability, MTT-assay was performed. The synthesized compounds (2a-f) were able to reduce DL cell viability in a dose dependent manner as compared to control cells (Fig. 1a and 1b). After 48 hr of incubation, the synthesized compounds (2a-f) were found to be cytotoxic to DL cells at concentration 200 µg/ml of compounds (2a, 2e, and 2f) and100 µg/ml of compounds (2b, 2c and 2d). The synthesized compounds (2a-f) reduced the cell viability to more than 50% of the initial level and this was used as the IC₅₀. In addition, prolonged exposures resulted in further increase toxicity to the cells. These results demonstrate that the synthesized compounds (2a-f) a concentration- and time-dependent increase in DL cell toxicity. Further, 200 µg/ml of compounds (2a, 2e, and 2f) and100 µg/ml of compounds (2b, 2c and 2d) were found to be the IC₅₀. Furthermore, experiments were carried out using these concentrations, to study the effect of the compounds (2a-f) on DL cells. It was found that cells treated with 200 µg/ml of compounds (2a, 2e and 2f) and 100 µg/ml of compounds (2b, 2c and 2d) for 12 hr, 18 hr and 24 hr showed significantly reduce cell viability as compared to control cells (Figure 1c and d). However, this reduction in viable cells was observed highly significant after 18 hr and 24 hr (Fig. 1c and 1d). In addition, the effect of the compounds (2a-f) was also evaluated on normal cells which suggested that normal cells did not affect at this concentration (200 µg/ml of compounds (2a, 2e and 2f) and 100 µg/ml of compounds (2b, 2c and 2d)) but required a high concentration to achieve IC₅₀. These findings suggest that DL cells showed sensitivity to the synthesized compounds (2a-f).

 

Figure 1 The effect of the synthesized compounds (2a-f) on the cell viability.

 

Effect of the compounds (2a-f) on DL cells morphology:

Next, to examine the effect of synthesized compounds (2a-f) on the cell morphology, Haematoxilin/Eosin, EtBr/Acridine and Hoechst-3342 staining were carried out. Results show that cells treated with 200 µg/ml of compound (2a, 2e and 2f) and 100 µg/ml of compound (2b, 2c and 2d) for 12 hr, 18 hr and 24 hr showed remarkable morphological changes at the levels of membrane and nucleus include membrane blebbing and bulging and acentric nuclei as compared to control (Fig. 2). However, these changes were observed highly prominent after 18 h than those of control cells (Fig. 2).  Therefore, these observations are corroboration with previous co-workers who suggest that tumor cells showed sensitivity to the chemotherapeutic compounds.

To further visualize nuclear morphology, treated cells were stained with Hoechst-33342 (2’-[4-ethoxyphenyl-1-1piperazinyl]-2,5’-bi-1benzimidazole trihydrochloride trihydrate), binds preferentially to A-T rich regions in the genetic materials. It was observed that incubation of DL cells with 200 µg/ml of compounds (2a, 2e and 2f) and 100 µg/ml of compounds (2b, 2c and 2d) for 12 hr, 18 hr and 24 hr results in disintegration/condensation of chromatin as compared to control cells (Fig. 3). Hoechst-3342 binds in minor groove of DNA and exhibits distinct fluorescence emissions at 460-490 nm in dye: base ratio dependent manner (Fig. 3). Compounds 2b and 2e showed marked changes in nuclear morphology after 18 hr; however, compounds 2a, 2c, 2d and 2f showed similar changes after 24 hr as compared to control cells (Fig. 3).

 

In order to confirm the previous findings, EtBr/acridine staining (a basic technique which stained DNA and cytoplasm) was carried out. The staining discriminated cells into three different populations; first, viable cells excluded EtBr but permeable to acridine orange, which intercalated into DNA to produced green fluorescent nuclei (Fig. 4). Second population, cells that had yellow chromatin in condensed nuclei and often had membrane blebbling, bulging and fragmented DNA were considered as apoptotic (Fig. 4). Third but not important is necrotic cells population showed collapse membrane integrity. To further clarify the facts, percent of apoptotic cells was determined with Alpha imager 2200 software by manual count (Fig. 5). It was observed that percent apoptotic cells was found increase after 12 hr, 18 hr and 24 hr as compared to control cells but this increase was observed highly significant after 18 hr and 24 hr respectively (Fig. 5). In contrast to percent apoptotic, the percent necrotic cells were found insignificant in all respective groups (Fig. 5).

 

Figure 2 Effect of the compounds (2a-f) on the DL cells morphology

 

Figure 3: Effect of the compounds (2a-f) on nuclear morphology in DL cells

 

 

Figure 4: Effect of the compounds (2a-f) on the morphology of DL cells

 

 

Figure 5 Effect of the compounds (2a-f) on apoptosis and necrosis

Effect of the compounds (2a-f) on DNA fragmentation and apoptosis of DL cells:

Apoptosis was further confirmed on molecular ground, therefore DNA fragmentation assay was carried out. It was observed that cells treated with the compounds (2a-f) for 12 hr, 18 hr and 24 hr results in appearance of low molecular weight DNA fragments (below 300 bp) as compared to control (Fig. 5). The effect of the compounds (2a-f) was observed more prominent after 18 hr and 24 hr as compared 12 hr (Fig. 6). These results are in agreement with previous findings which suggest that the compounds (2a-f) were able to induce morphological changes (membrane blebbing, bulging and nuclear condensation), DNA fragmentation/degradation and apoptosis in DL cells.

 

Figure 6 Effect of the compounds (2a-f) on DNA fragmentation and apoptosis.

 

DISCUSSION:

The present report demonstrates that the synthesized quinoxaline derivatives (2a-f) inhibited cell proliferation, altered cell morphology and induced DNA fragmentation and thus apoptosis in DL cells. Treatment with the compounds (2a-f) results in reduced cell viability, increase cytotoxicity, induced morphological alterations (plasma membrane blebbing, nuclear disintegration) and fragmentation of DNA into low molecular weight DNA fragments and apoptosis. Therefore, the findings are in agreement that the studied compounds (2a-f) showed remarkable therapeutic potential against Dalton’s lymphoma cells, however additional studies are needed to decipher the underlie mechanism in other animal models.

 

The compounds (2a-f) inhibited DL cell growth and proliferation in concentration- and time-dependent manner. These findings are in agreement with the previous studies which suggest that quinoxaline derivatives showed anti-cancer and anti-inflammatory activities against several kind of tumor. Further, studies show that cells were more sensitive to the compounds (2a-f) which results in membrane blebbing, bulging and nuclear disintegration in DL cells. In addition, treatment with the compounds (2a-f) for 18 hr and 24 hr showed a significant decrease in the number of proliferating cells as compared to untreated cells. The decrease in proliferating cells was considered corresponding to the number of apoptotic cells. Among all, the compounds (2b and 2e) showed significant anti-tumor/anti-proliferative effect against this cell line as compared to others. Moreover, the toxicity induced by the compounds (2a, 2c, 2d and 2f) was observed significantly higher as compared to control cells. This may be due to the presence of different substituent in quinoxaline derivatives (2a-f) by which they exert different effects on DL cells. Therefore, inhibition of cell proliferation by the quinoxaline derivatives (2a-f) resulted in altered cell morphology such as acentric nuclei, membrane blebbing and bulging, nuclear disintegration, chromosomal condensation.

 

Furthermore, the susceptibility of DL cells to the compounds (2a-f) might be due to their DNA binding ability. Although, the synthesized compounds (2a-f) belong to the same class but they have different substituent and this may be a cause to show different DNA binding ability of the compounds (2a-f) in DL cells. Therefore, treatment with the compounds (2a-f) results in fragmentation of genomic DNA into low molecular weight DNA fragments (below 300bp). Thus, appearance of these low molecular weight DNA fragments is the confirmation of apoptosis in DL cells. However, normal spleenocyte cells did not show any cytotoxicity or apoptosis at this IC₅₀ of the compounds (2a-f).

 

CONCLUSION:

Taking together these findings, it might be concluded that treatment the compounds (2a-f) reduced cell viability in concentration- and time-dependent manner, induced morphological changes (membrane blebbing, bulging and nuclear disintegration) and fragmentation of genomic DNA into short DNA fragments and subsequent apoptosis in DL cells. Therefore, the synthesized compounds (2a-f) may be better therapeutic regimen to treat/eliminate this type of cancer without affecting the normal cells. However, additional studies are needed to uncover the different facets of the compounds (2a-f). The challenges for future studies will improve the understanding of readers and researchers in this area.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGEMENT:

Authors are thankful to Prof. Arbind Acharya and Sanjay Kumar, Department of Zoology, Banaras Hindu University for providing necessary lab support during this investigation. 

 

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Accepted on 12.01.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3165-3171.