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ORIGINAL ARTICLE
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BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins


1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
2 Department of Physiology, College of Medicine, King Khalid University, Abha, Saudi Arabia
3 Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi, India
4 Department of Basic Medical Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
5 Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
6 Department of Zoology and Environmental Sciences, GKV, Haridwar, India
7 Department of Pharmaceutics, Era College of Pharmacy, Era University, Lucknow, UP, India

Date of Submission04-Sep-2020
Date of Decision20-Dec-2020
Date of Acceptance03-Jan-2021
Date of Web Publication18-Aug-2021

Correspondence Address:
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1281_20

 > Abstract 


Objective: The present study aimed to investigate the inhibitory role of second mitochondria determined activator of caspases mimetic on inhibitor of apoptosis proteins (IAPs) and regulation of caspases in nonsmall cell lung cancer cell line.
Materials and Methods: Dimethyl sulfoxide and 3-(4, 5-dimethyl thizol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was done to determine the IC50 of BV6 using NCI-H23 cell line. The levels of mRNA of X-linked IAP (XIAP), cellular IAP (cIAP-1), cIAP-2, caspase-6, and caspase-7 in H23 cell line were evaluated by a quantitative real-time polymerase chain reaction, while their protein expressions were tested using western blotting.
Results: Two doses of BV6 dependently downregulated the expression of mRNA of XIAP (P = 0.002, P= 0.0003 vs. untreated), cIAP-1 (P = 0.05, P = 0.005 vs. untreated), and cIAP-2 (P = 0.001, P = 0.0002 vs. untreated), respectively, while the compound upregulated the mRNA expression of caspase-6 (P = 0.001, P < 0.0001 vs. untreated) and caspase-7 (P = 0.001, P = 0.0004 vs. untreated), respectively. Dose dependent of BV6 treatment significantly decreased the protein level of XIAP (P = 0.003, P = 0.007 vs. untreated), cIAP-1 (P = 0.02, P = 0.01 vs. untreated), and cIAP-2 (P = 0.008,P = 0.008 vs. untreated), respectively. However, the compound increased the protein level of caspase-6 and caspase-7 when compared to untreated control (P = 0.006,P = 0.001) and (P = 0.01, P = 0.001), respectively.
Conclusions: The result showed that BV6 treatment reduced the level of mRNA of XIAP, cIAP-1, and cIAP-2 and increased the gene expression of caspase-6 and caspase-7 in NCI-H23 cell line. Therefore, the study revealed that BV6 could be used in future as additional therapeutics in lung cancer.

Keywords: BV6, caspase, cellular inhibitor of apoptosis proteins -1, cellular inhibitor of apoptosis proteins -2, lung cancer, X-linked inhibitor of apoptosis proteins



How to cite this URL:
Ahmad I, Dera AA, Irfan S, Rajagopalan P, Ali Beg MM, Alshahrani MY, Mir MA, Abohashrh M, Alam MM, Wahab S, Verma AK, Srivastava S. BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=324031




 > Introduction Top


Malignancy of the lung remains as a predominant reason for disease-related mortality worldwide.[1] Of the diagnosed cases, 85% of lung cancer cases were analyzed as nonsmall cell lung carcinoma (NSCLC) where chemotherapy remains as the leading treatment to tackle this dreadful form of disease.[1] Despite the recent advances in chemotherapy regimen, it is estimated that 5-year survival rate of NSCLC patients is only 15%, mainly due to the drug resistance.[2] Selective induction of apoptosis has been promising focus on the advancement of new treatment techniques that may prompt upgrade the chemo- and radio-affectability of the malignant cells.[3] Characteristic and extraneous apoptotic pathways have been considered as the main two distinctive apoptotic pathways. Both intrinsic and extrinsic pathways involve the activation of caspases-6 and caspase-7, which leads to the fragmentation of DNA and causes cell death.[4]

Recent studies have thrown light on investigating any anticancer molecule at the molecular level to control the rapidly dividing cancerous cells.[5] Inhibitor of apoptosis proteins (IAPs) is a family of proteins that has recently gained momentum in cancer research.[6] X-linked IAP (XIAP) is one such IAP mainly associated with inhibition of both initiator and effector caspases.[7],[8] Further, XIAP, cellular IAP (cIAP-1), and cIAP-2 are a compelling inhibitor of apoptosis, while clinical data indicate a correlation with upregulation of these proteins with poor prognosis and advancement of the disease.[9] Studies indicate IAPs to exercise their antiapoptotic conduct through direct restraint of initiator and effector caspases.[10] IAPs have also been specifically involved in ubiquitination of caspase proteins, ultimately leading to inhibition of apoptosis. However, cIAP1 and cIAP2 regulate antiapoptotic activity by shedding of tumor necrosis factor-alpha-mediated nuclear factor kappa B signaling.[10] Overexpression of IAPs constitutively promotes malignancy by inhibiting apoptosis.

Second mitochondria determined activator of caspases (SMAC)/DIABLO direct IAP-restricting protein with low isoelectric point (PI) is reported as an important contradictor of IAP-intervened caspase restraint in human cells.[11] Decreased expression of SMAC has been observed in lung cancer, and lower expression was associated with worse disease prognosis.[12] SMAC mimetic compounds have been synthesized to target multiple IAPs, including cIAP1, cIAP2, and XIAP in cancer treatment.[13] BV6 is bivalent IAP antagonists, which has been shown to involve in proteasomal degradation of cIAP1 and cIAP2, an ultimately promoting apoptosis and cell death.[14] To the best of our knowledge, the effect of BV6 in NSCLC cell line remains elusive. Therefore, the present study aimed to investigate the role of SMAC mimetic in the regulation of IAPs and caspases in nonsmall cell lung cancer cell line.


 > Materials and Methods Top


Materials

All the chemicals and reagents were purchased from Sigma (St. Louis, MO). NCI-H23 cell line was purchased from the American Type Culture Collection. Western blot antibodies were purchased from Abcam (Cambridge, UK). RNA was isolated using through all untreated and treated cell lines using Trizol (invitrogen) reagent. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) reactions were carried out using SYBR Green I technology. SMAC mimetic compound BV6 was purchased from Sigma-Aldrich, USA.

Cell culture

Cells were cultured in RPMI-1640 media supplemented with 10% of fetal cow-like serum fetal bovine serum, 0.01 mg/mL of insulin, 100 U/mL of penicillin, 100 μg/mL of streptomycin, 250 ng/mL of amphotericin, and 250 μg/mL of gentamicin. Cells were kept up at 37°C in 5% CO2 hatchery containing sticky air. The medium was replaced every alternate day and maintenance was strictly followed in accordance with standard methods. Assays were performed when the cells were ≤70% confluent.

Methods

Cell viability assay

MTT assay was performed to calculate IC50 value for SMAC mimetic compound BV6 (Sigma-Aldrich, USA) and the calculated IC50 value of BV6 was 20 μM.[15] Two treatment doses (1/20 and 1/10 of IC50) of BV6 such as 1 μM and 2 μM were selected. Untreated control group was also taken and 48 h of treatment was given to NCI-H23 cell lines and the cells were further harvested to extract the total RNA and protein as well as normal lung cell line Medical Research Council cell strain-7 (MRC-7) was taken as a reference for mRNA expression calculation in untreated NCI-H23 cell lines.

Total RNA extraction, cDNA synthesis

The extraction of RNA was done through all untreated and treated cell lines using Trizol (Invitrogen) reagent as per the instruction given by manufacturers and stored at −80°C until additional necessary step for cDNA synthesis. RNA concentrations and purity were measured using a Thermo Scientific NanoDrop™ 2000 spectrophotometer. Hundred nanogram of total RNA, without reverse transcriptase and DNA controls, was used to synthesize cDNA using (Verso, Thermo scientific, USA) following the manufacturer's protocol.

Quantitation of mRNA levels using real-time quantitative polymerase chain reaction

Twenty microliter samples were subjected to RT-qPCR for XIAP, cIAP-1, cIAP-2, caspase-6, and caspase-7 and β-actin using SYBR Green I technology using the primers indicated in [Table 1]. XIAP, cIAP-1, cIAP-2, caspase-6, and caspase-7 mRNA expression level study was executed by using the program for 40 cycles, first denaturation step at 94°C for the 40s, annealing was for 20s at 60°C, and extension was carried out at 72°C for 30s. The final step for extension was at 72°C for 10 min. Melting curve analysis was done between the temperature ranges of 35°C–90°C to make sure the target amplification and all the reactions were performed in duplicate. The relative levels of each RNA were determined from the Ct value after normalization with control mRNA, β-actin using a 2−(ΔΔCT).[16] The results were shown as mean fold change in the cell lines compared to counterpart.
Table 1: Primer sequences for X-linked inhibitor of apoptosis, c inhibitor of apoptosis -1, c inhibitor of apoptosis -2, caspase-6, and caspase-7 mRNA and β-actin

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Western blot analysis for inhibitor of apoptosis proteins, caspase-6, and caspase-7

All the cells were lysed in lysis buffer, and the total protein concentration was estimated by Coomassie Plus Protein Assay Reagent kit (ThermoFisher, USA). Thirty microgram proteins from the cell lysate were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane film by semi-dry blotting method (Merck, New Jersey, USA) probed with respect to the primary antibodies for 15 h at 4°C. Horseradish peroxidase secondary antibodies were added and the membrane was stripped after 3 h of an incubation at 25°C. Bands were estimated in ChemiDoc XRS + by utilizing Image Lab programming (Bio-Rad) and standardized with β-actin (1:5000).

Statistical analysis

All the data analyses were performed using SPSS 20.0 and GraphPad Prism 5.03 (La Jolla, San Diego, California, USA). Expressions of XIAP, cIAP-1, cIAP-2, caspase-6, and caspase-7 were presented as mean + standard deviation, while the protein band density was observed by Image Lab software (Bio-Rad). Based on the outcomes of parametric t-test, analysis of variance and nonparametric Mann–Whitney U-test and Kruskal–Wallis tests were performed to see the statistical differences among the different groups and P < 0.05 was considered to be statistically significant.


 > Results Top


BV6 inhibited lung cancer cell proliferation MTT assay was implemented to analyze the IC50 value of SMAC mimetic compound on NCI-H23 cell line. To examine the cytotoxicity of BV6, NCI-H23 cells were treated with different doses of BV6 (2.5μM to 25μM) for 48 h [Figure 1]. We used the respective 1/20 and 1/10 of IC50 such as 1 μM and 2 μM of BV6 in NCI-H23 cell line for testing the dose dependency of BV6 for the following assays.
Figure 1: BV6 induces cytotoxic effect in NCI-H23 lung cancer cell line. BV6 induced cytotoxicity in NCI-H23 cells as confirmed by MTT assay, cell survival was measured with MTT assays with specified concentrations of BV6 for 48 h. The results are represented as means ± standard deviation of three independent experiments. Nonsignificant (ns), *P < 0.05, **P < 0.01 versus untreated control

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BV-6 inhibited the expression of XIAP, cIAP-1, and cIAP-2 mRNA, while it increased the expression of caspase-6 and caspase-7 mRNA.

Treatment of BV6 showed decreased XIAP, cIAP-1, and cIAP-2 mRNA expressions compared to the untreated control group [Figure 2]a, [Figure 2]b, [Figure 2]c. Untreated control group showed 8.97 ± 0.72 fold for XIAP mRNA expression, while the cells treated with 1 μM and 2 μM decreased the expression of XIAP mRNA significantly (4.19 ± 0.69, 2.61 ± 0.53 folds; P = 0.002, P = 0.0003 respectively). Similarly, the relative levels of cIAP-1 mRNAs in untreated control group showed 8.05 ± 0.96 fold, while BV6 treatment significantly reduced cIAP-1 mRNAs (3.13 ± 0.72 fold P = 0.05 for treating with 1 μM, 1.93 ± 0.13 P = 0.005 with 2 μM). Likewise, cIAP-2 mRNA expression was decreased after treated with 1 μM of BV6 for 3.69 ± 0.89 fold (P = 0.002) and 2 μM of BV6 for 2.84 ± 0.15 (P = 0.0001) when compared to the untreated control group which showed 7.83 ± 0.54-fold mRNA expression.
Figure 2: Relative levels of X-linked inhibitor of apoptosis proteins, cellular inhibitor of apoptosis proteins-1, cellular inhibitor of apoptosis proteins-2, caspase-6, and caspase-7 mRNA expression in NCI-H23 cell line by RT-qPCR. Levels of mRNAs encoding X-linked inhibitor of apoptosis proteins (a), cellular inhibitor of apoptosis proteins -1(b), cellular inhibitor of apoptosis proteins -2 (c), caspase-6 (d), and caspase-7 (e) shows a statistically significant difference in the levels of each mRNA after treating with 1 and 2 μM of BV6 once comparing with the untreated control. After normalizing to actin, the results from three individual experiments were expressed as mean ± standard deviation and were plotted

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However, BV6 showed an increase in the expression of caspase-6 and caspase-7 mRNA once compared with untreated control. The level of caspase-6 mRNA in the untreated control group was 0.40 ± 0.11 fold, while treated with 1 μM and 2 μM, it increased the expression of caspase-6 mRNA significantly, as illustrated in [Figure 2]d (1.62 ± 0.26 fold; P = 0.001 and 3.43 ± 0.20 fold; P < 0.0001, respectively). Similarly, an increase of caspase-7 mRNA expression was observed in the treated groups [Figure 2]e, the untreated control group showed 0.31 ± 0.07-fold caspase-7 mRNA expression, while treated with 1 μM and 2 μM, it showed 2.92 ± 0.59-fold and 3.22 ± 0.44-fold caspase-7 mRNA expression, respectively (P = 0.001, P = 0.0004).

BV-6 treatment and XIAP, cIAP-1, cIAP-2, caspase-6, and caspase-7 protein expression.

Treatment of BV6 showed a significant decreased in the level of XIAP, cIAP-1, and cIAP-2 protein expressions compared to the untreated control group [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d. BV6 treatment with 1 μM and 2 μM showed a significant reduction in XIAP protein expression compared to untreated control (P = 0.003, P = 0.007, respectively). It was also observed that the expression of cIAP-1 protein in the treated with 1 μM and 2 μM of BV6 was significantly reduced when compared to untreated control (P = 0.02, P = 0.01, respectively). The protein expression of cIAP-2 was decreased once treated with 1 μM and 2 μM of BV6 after comparing to untreated control (P = 0.008, P = 0.008, respectively).
Figure 3: Levels of X-linked inhibitor of apoptosis proteins, cellular inhibitor of apoptosis proteins-1, cellular inhibitor of apoptosis proteins-2, caspase-6, and caspase-7 protein expression in NCI-H23 by western blotting. (a) Western blot of antiapoptotic markers after various doses of BV6 treatment. Levels of X-linked inhibitor of apoptosis proteins (b), cellular inhibitor of apoptosis proteins -1(c), cellular inhibitor of apoptosis proteins -2(d), caspase-6 (e), and caspase-7 (f) proteins show a statistically significant difference in the levels of each protein after treating with 1 and 2 μM of BV6 once comparing with the untreated control. After normalizing to actin, the results from three individual experiments were expressed as mean ± standard deviation and were plotted

Click here to view


In the contrast, BV6 showed an increase in the caspase-6 and caspase-7 protein expression once compared with the untreated control [Figure 3]e and [Figure 3]f. The level of caspase-6 protein was increased in the cells treated with 1 μM and 2 μM of BV6 compared to untreated control (P = 0.006, P = 0.001, respectively). An increased level of caspase-7 protein was also observed in the cells treated with 1 μM and 2 μM of BV6 compared to untreated control (P = 0.01, P = 0.001, respectively).


 > Discussion Top


Nonsmall cell lung disease has been dominatingly analyzed in the cutting edge stage, and treatment regularly requires radiotherapy in blend with other treatment modalities, for example, chemotherapy. Nevertheless, the combined 5-year survival time is only 15%. In spite of the ongoing progression in the treatment of cancer, it was demonstrated that alterations in the apoptotic pathways of the carcinoma cells play a role in the drug resistance.[2]

Targeting apoptosis pathways has been a promising target in development, improvement, and sensitivity to current therapy of cancer cells. Bivalent IAP antagonists BV6 have been appeared to actuate proteasomal degradation of cIAP1 and cIAP2 at last prompts caspases instigated apoptosis of disease cells.[14] SMAC is the natural antagonist of IAPs present in the cells, naturally released during apoptosis from mitochondria[17] and mechanistically showed interaction with N-terminus of SMAC with the surface of the IAP proteins.[18] The development of SMAC mimetics has been used exogenously to target the IAPs in cancer research.[18] Bivalent SMAC mimetics have been viewed as 100–1000 times more impressive than monovalent mimetic mixes in inciting the apoptosis of carcinoma cells. Bivalent SMAC mimetics incorporate their improved capacity to repeal XIAP intervened caspase hindrance.[19] BV6 is a bivalent SMAC mimetic and has recently been appeared to estrange IAP and trigger proteasomal corruption of IAP proteins.[20] In this study, we have shown that 1 μM and 2 μM of BV6 treatment successfully have more than 4- and 6-fold reductions in XIAP and cIAP-2 mRNA expression compared to untreated control NCI-H23 cell line. Dose dependent of BV6 treatment showed more than 1–3-fold increase in caspase-6 and caspase-7 mRNA expression compared to the untreated control cell. Apoptosis proteins (IAPs) are exceptionally communicated in different tumors including cervical malignant growth, esophageal squamous cell carcinoma, hepatocarcinoma, medulloblastoma, a few types of lung disease, pancreatic disease, and others.[21],[22] Therefore, IAPs can be used to potentially indulge in cancer treatment. Various SMAC mimetics have been tested and under the development to target IAPs as new cancer treatment alternatives.[20] BV6 targets cIAP1, cIAP2, and XIAP, which square apoptosis by straightforwardly repressing caspases by activating the pro-survival mechanism.[20] BV6 induces the apoptosis by blocking the IAPs in tumor cells. It has been suggested that SMAC mimetic stimulates the necroptosis in some settings, which could be another mode of cell death.[23]

The current study showed that the treatment of BV6 with 1 μM and 2 μM had a significant impact on the reduction in XIAP, cIAP-1, and cIAP-2 protein expression, while increased caspase-6 and caspase-7 protein expression was observed. This is indeed suggested that inhibition of IAPs leads to increased expression of caspases. Hofmann et al. revealed that the XIAP involved in the NSCLC pathogenesis,[24] as well as Berezovskaya et al. recommended that the XIAP participated in metastasis in prostate cancer patients.[25] Several studies revealed that high expression levels of XIAP in cancer predicted a worse prognosis and decreased median survival of patients. Recently, it has been suggested that XIAP can be an attractive target in the development of a new cancer treatment line.[26] Small molecules like SMAC as XIAP inhibitors have been involved in the inhibition of cancer cells with low toxicity.[27] The research on cancer in the last decade focused on developing new drugs by targeting XIAP and involving in suppressing cancer growth and enhancing chemotherapy sensitivity has been revealed in several cancers.[28]

It has been observed that the increased expression of XIAP was observed in ovarian carcinoma cells during the treatment.[29] High-affinity SMAC mimetic can be used to target IAPs that may be the potential tool to control cancer growth.[30] It was suggested that the downregulation of XIAP protein by SMAC could enhance apoptosis and increases the chemosensitivities in gastric cancer cells. Current evidence revealed that caspases are the essential machinery of the cell death process.[31] It has been revealed that SMAC mimetics could be the potential therapeutic agents to increase the antitumor action of proteasome inhibitors such as carfilzomib (CFZ), ixazomib, and oprozomib together.[32]

A study by Anna et al. in 2020 shown that SMAC mimetics and proteasome inhibitors represent a favorable treatment approach for primary diffuse large B-cell lymphoma (DLBCL), and experiments presented that BV6 and CFZ supportively induce cell death via the mitochondrial pathway proposed SMAC mimetics for sensitizing DLBCL cells.[33]

SMAC can stimulate the degradation of cIAPs and several cancer overexpress IAPs, consequently enabling tumor cells to evade apoptosis, and SMAC mimetics can target tumors with high levels of cIAPs.[34] In a study by Ji et al. on lung cancer cell lines A549 and H460, the result specified that SirT1 regulates apoptosis and radiation sensitization in lung cancer cell lines A549 and H460 via the SMAC pathway.[35] In another study, it is suggested that SMAC could be used to elicit the apoptosis probability by noncanonical cell death pathways when applied at high concentrations.[36]

A study indicated that BV6 induced cell death in different human cancer cell lines and revealed that BV6 dose dependently induced deprivation of IAPs including cIAP1 and cIAP2, targeting of IAPs by BV6 might be a promising approach to reduce the cancer growth.[37]


 > Conclusions Top


The study concluded that SMAC mimetic BV-6 potentially targets the IAPs and increased the gene expression of caspase-6 and caspase-7 in the NCI-H23 cell line. This could be used or indulge as a potential therapeutic agent in NSCLC treatment and management. Data suggested that SMAC mimetic BV6 could be used as a potential new therapeutic approach in lung cancer and can help to overcome the problem of therapeutic resistance.

Acknowledgments

The authors are thankful to the Deanship of Scientific Research, King Khalid University, Abha, Saudi Arabia, for financially supporting this work through the General Research Project under grant number (R. G. P. 1/159/40).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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    Figures

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    Tables

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