TRAIL-resistant glioma cells resist Withania somnifera mediated apoptosis via USP5 upregulation

Abstract

The tumor necrosis factor related apoptosis inducing ligand (TRAIL) shows a potential therapeutic by inducing apoptosis in glioma cells and sparing normal cells, but TRAIL resistance and chemotherapeutic resistance still prevails in glioma and hinder its apoptotic effect. To address TRAIL resistance, we investigated the potential of Withania somnifera, a medicinal herb with recognized anti-cancer properties. The response against the therapeutic approaches led glioma to get regress but relapse is a major challenge. The study aims to evaluate the biological effect of the W. somnifera fruit extract in TRAIL resistant glioma cell lines (U87MG, LN229) and TRAIL sensitive (T98G) glioma cell lines. MTT cell viability assay were performed to assess the effect of W. somnifera fruit extract. Fluorophore conjugated Annexin V APC -PI staining was performed to study the apoptotic effect of an extract obtained from W. somnifera. The underlying mechanism comprises of USP5 mediated DR5 regulation in cell survival and its subsequent knock down led to apoptosis enhancement. The apoptotic relevant proteins SMAC, and semi quantitative PCR analyzed abundance of DR4 & DR5 receptor was quantitated. Therefore, the expression of USP5 was analyzed across 592 glioma tumor data and was correlated with the abundance of EGFR, the data was accessed from cBioportal (https://www.cbioportal.org/). Our study findings proposed the leading upregulation of deubiquitinating enzyme USP5 in TRAIL resistant U87MG and LN229 glioma cells upon treatment with Withania somnifera fruit extract promotes cell survival. Furthermore, depletion of USP5 followed with Withania somnifera fruit extract-treatment, upregulates TRAIL receptor (DR5) and SMAC protein led to apoptosis activation, reveals a survival promoting characteristic of Withania somnifera fruit extract in TRAIL resistant U87 MG and LN229 glioma cells. Furthermore, being USP5 a key promoter in tumorigenesis but is found overexpressed/mutated in 3% of glioma tumor. The leading upregulation of USP5 found interesting in our treatment set to correlate with the EGFR abundance across the tumor samples. Since, EGFR is overexpressed in 47% tumor and acquiring the potential chemo-resistant pathway led to USP5 upregulation may be detrimental causing recurrent tumor. Therefore, from our studies USP5 deubiquitinating enzyme plays a key role in latent survival. Deubiquitinating enzyme family protein USP5 upregulation and stability of apoptotic protein SMAC are found important indicator in balancing the TRAIL resistance glioma to opt unidirectional pathway which is not aberrant but rewired on W. somnifera FE treatment. Therefore, knockdown of USP5 make cells sensitive to undergo direct apoptosis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-00917-3.

Introduction

Glioblastoma is an aggressive metastatic cancer with a median patient survival of around one year, remaining life-threatening due to its recurrence phenotype^1,2. In normal cells, maintaining orderly regulated cellular protein homeostasis via the protein ubiquitination pathway ensures health or turn down to disease³. In cancerous cells, the disruption in protein ubiquitination leads to its homeostasis failure causing glioma⁴. The ubiquitin machinery is comprised of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) that controls substrate specificity for protein ubiquitination, apparently deubiquitinating enzymes(DUB) family proteins, remove ubiquitin moieties from substrate proteins and plays a crucial role in balancing protein half-life⁵. Uncontrolled action of DUB family enzymes such as USP7, USP14, USP21, and USP13 regulates cell proliferation and apoptosis in several cancers including gliomas^6–8. One such DUB from the ubiquitin-specific protease family of DUBs is USP5, also known as isopeptidase T, which has several targets that maintain the ubiquitin pool inside the cytosol⁹. Emerging shreds of evidence suggest that USP5 expression is increased in glioma tissues compared to normal brain tissues, and plays a critical role in glioma genesis by modulating the ubiquitination status of proteins involved in tumor growth, invasion, and therapy resistance¹⁰. USP5 stabilizes cyclin D1 through direct interaction and promotes tumorigenesis and cell proliferation in glioma¹⁰. USP5 also help in maintaining the invasiveness properties of mesenchymal glioma stem cells through stabilization of OCT4 and promotes tumorigenesis¹¹. On the other hand, anti-tumorigenic ligands also play a key role in prohibiting cancer cell formation, where tumor cells can resist cell death by attenuating extrinsic apoptotic pathway via cell death receptors in the presence of FAS, TRAIL, TNF- alpha¹². Among these, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) belongs to the TNF family of proteins¹³. TRAIL is notable for its ability to induce apoptotic cell death specifically over unwanted cell growth, while sparing normal cells^14,15. TRAIL ligand reportedly induces apoptosis through its transmembrane TRAIL receptors DR4 and DR5, apart from DR4 and DR5 there are several other TRAIL receptors (DcR1, DcR2 and osteoprotegerin) which aids in survival signaling^16–18. When the TRAIL ligand binds to DR4/DR5 receptors, it forms a homo-trimeric complex and employs its intracellular death domain (DD) to recruit FADD (Fas-associated death domain) and caspase 8, collectively forming DISC complex mediates apoptosis¹⁹. Although TRAIL ligands are highly expressed in tumor cells, but can easily escape apoptosis by expressing specific ubiquitin enzymes like TNFAIP3²⁰. Knockdown of TNFAIP3 induces TRAIL-mediated apoptosis by inhibiting RIP1 ubiquitination, which leads to caspase 8 activation in hepatocellular carcinoma²¹. Interestingly, the knockdown of another deubiquitinating enzyme, USP5, in melanoma resulted in FAS protein downregulation, suggesting that USP5 may have a role in the extrinsic pathway of apoptosis²².

Previous studies suggested that glioma cells are also resistant to TRAIL-mediated apoptosis²³. Interestingly, TRAIL ligand and FAS ligand cleave USP5 in TRAIL-sensitive glioma cells and induce apoptosis but no such cleavage or apoptosis was observed in TRAIL resistance glioma cells²⁴. Thus, USP5 has a significant role in inducing apoptosis in gliomas. Various chemotherapeutic drugs in combination with TRAIL ligands are used to induce apoptosis in TRAIL resistance cells by targeting caspase 8 or inhibitory c-FLIP, additionally targeting deubiquitinating enzyme USP5 also could be of more therapeutic use^25,26, if is the mediator in apoptosis. Keeping these important signal mediators to decipher the resistance complexity using Withania somnifera fruit with anti-cancerous benefits has been scaled out in the present study. Since, Withania somnifera commonly known as Ashwagandha, was been used majorly as a traditional medicine in use throughout the Asia and Europe for treating various ailments and also in anti-cancerous use^27,28. Different groups studied Withania somnifera also has anti-cancerous properties, focused mainly on its root extract which has bioactive compounds like withanolides and withaferin A, that can induce apoptosis, and suppress cancer invasiveness. Recently, various important medicinal herbs based compounds including W. somnifera crude generic extract is under major attention in promoting more toxicity²⁹, though such medicinal herbs been used widely in home globally causing non clarity in determining the outcome of targeted based therapy in treating various metabolic diseases. Lack of predictive overall survival of cancer patient in prescribed chemotherapy along with non-prescribed medicinal herbs deviate the real finding to cure the disease or relapse in the disease if is cancer.

In our study, we as per procedure made a chloroform and methanol solvent mediated extract formulation of Withania somnifera fruit in presence of TRAIL ligand. This to study its effect on TRAIL ligand sensitive (T98G), and TRAIL resistant (U87 MG, and LN229) glioma cell lines. In TRAIL resistant glioma cells U87 MG and LN229, we have notably studied the USP5 upregulated expression upon treatment with Withania somnifera.

Material and method

Cell culture

Glioma Cell line T98G (Generous support from Prof. Kunzang Chosdol, AIIMS, New Delhi(India)), U87 MG, and LN229 (NCCS, Pune India) were grown in Dulbecco’s modified Eagle’s medium (HiMedia) supplemented with 10% fetal bovine serum(HiMedia) and 100units of Penicillin-Streptomycin (Gibco). Cells were grown inside a 5% CO₂, humidified chamber at 37^◦C.

Collection of plant material

The plant material was collected from New Delhi, India, and was submitted for identification to taxonomist – Dr. Shruti Kasana, Department of Botany, University of Delhi, Delhi India. The reference number DUH14865 has been retained in the concerned herbarium of the University of Delhi.

Withania somnifera solvent preparation strategy

Withania somnifera methanol extract from fruit was prepared with some modifications³⁰. The dried fruit from the plant was collected in the early morning and further sun-dried to remove all the moisture. The completely dried fruits were weighed around 0.84 g followed by give initial wash with 70% Ethanol to remove any microbial contamination over the fruit and were later completely air dried and powdered using a pestle motor and suspended in 1 ml of 100% methanol. The mixture was vortexed for 5 min, followed by sonication for 15 min, and then placed in a shaker for 1 h at room temperature. The complete mixture was centrifuged at 13,000 rpm for 5 min and supernatant was collected. The same steps were performed thrice with 100% methanol. After collecting the methanol extract of Withania somnifera fruit, it was kept at 60^◦C for 2 h in Rota vapor and pretreated with chloroform for 1 h twice and filtered using Whatman filter paper. The pretreatment of chloroform was performed to remove the lipids, fatty acids, and oil texture of the W. somnifera FE³¹. The chloroform non-soluble fraction was dried and was then dissolved in 100% methanol at a concentration of 20 mg/ml as our stock concentration and was filtered through a 0.22 μm syringe filter, before the cell treatment experiment. The HPLC was performed to analyze the presence of different compounds measured at 254nM (Supplementary Fig. 1). The subsequent experiments were performed in the methanol-dissolved, chloroform non-soluble fraction for the study.

Cell viability by MTT assay

The effect of W. somnifera FE and Recombinant human TRAIL protein on cell viability was measured by MTT assay. 1 × 10⁴ cells, T98G, and U87 MG glioma cells were seeded into 96 well-cell culture plates. After 24 h of incubation, cells were treated with W. somnifera FE at different concentrations of 100 µg/ml, 200 µg/ml, and 200 µg/ml and recombinant human TRAIL protein at 0.25 µg/ml, or in the combination of recombinant TRAIL 0.25 µg/ml with different concentration of W. somnifera FE for 48 h. Following treatment, the MTT reagent was added at 500 µg/ml and incubated for 2 h at 37ºC. After this, 150 µl of DMSO was added to dissolve the formazan crystals and again incubated for 20 min³². The absorbance was measured at 570 nm in an ELISA plate reader (TECAN).

siRNA transfection

T98G, U87 MG, and LN229 glioma cells (2 × 10⁴cells) were seeded and after 17 h of plating, cells were transfected with siRNA specific for USP5 (10nM) using Interferrin transfection reagent (Polyplus) as per protocol. USP5 sequence 5’- GAUGGGUGAUCUACAAUGA-3’. After 48 h of transfection, cells were treated with either TRAIL 0.25 µg/mL, W. somnifera FE 200 µg/mL, or in a combination of both for 24 h. Protein lysates were prepared in a cell lysis buffer (Cell signaling) containing protease inhibitors (Abcam) and phosphatase inhibitors (Sigma). siRNA-mediated knockdown efficiency was found ≥ 50–60%, as per western blot data.

Western blotting and antibodies

T98G and U87 MG glioma cells (2 × 10⁶) were seeded and after 24 h of plating cells were treated with TRAIL 0.25 µg/mL and W. somnifera FE 200 µg/mL either separately or in combination for 6 h. Following treatment, total protein lysates were made from SDS-cell lysis buffer (Cell Signaling Technology). The protein concentration was determined by the Bradford reagent(BioRad). The total protein of concentration 20 µg was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF (MDI) membrane at 20 V overnight at room temperature. The next day, membranes were blocked with 5% non-fat dry milk for non-phosphorylated proteins or blocked with 5% BSA for phosphorylated proteins for 1 h. After this, the membrane was washed using Tris-buffered saline with Tween 20 3 times (5 min each) and processed for specific primary antibodies overnight at 4º C. The primary antibodies against anti- USP5(Proteintech, 10473-1-AP, 1:10000), anti- LC3B (abcam, 1:2500), anti-XIAP (Abcam, ab28151, 1:2000), anti-SMAC (Abcam, ab32023, 1;2500), anti- BCL-2(Santa- Cruz biotech. Inc.-1:1000), USP8 (1:10000, 67321-1-Ig), and anti-β-actin (Biospes, BTL1027, 1:5000). Followed by probing with specific secondary antibody (1:5000) for 1 h and developed using Chemiluminescent reagent(Bio-Rad), under Chemiluminescent Gel Documentation system. Horseradish peroxide conjugated secondary antibodies (anti-mouse, GeNei, 114068001 A, and anti-Rabbit, GeNei, 114038001 A) were used.

Semi-quantitative PCR

T98G and U87 MG glioma cells were seeded 2 × 10⁶ and grown to 70–80% confluency for 24 h. After 24 h of plating cells were treated with TRAIL 0.25 µg/mL and W. somnifera FE 200 µg/mL either separately or in combination for 6 h and total mRNA was extracted using TRIsoln (GeNei). The RNA integrity and stability were assessed by running RNA on agarose gel electrophoresis further after checking integrity of RNA, the Nanodrop N-1000 instrument were used for RNA quantification followed by cDNA preparation using reverse transcriptase kit (Primescript 1st strand cDNA synthesis kit, Cat no.- 6110 A). 1ug of total RNA was subjected to cDNA preparation using a cDNA reverse transcriptase kit as per the manufacturer’s protocol. The list of Primers used for semi-quantitative PCR is in Table 1.

Table 1.

List of primers used for semi-quantitative PCR.

Sr. No.	Gene	Sequence	Denaturation	Annealing	Extension
1.	DR4 forward	Forward 5’- CTCAGCTGCAACCATCAAAC-3’ Reverse 5’- CAGGGACTTCTCTCTTCTTCATC-3’	95◦C for 30 s.	54◦C for 45 s	72◦C for 1 min
2.	DR5 forward	Forward 5’- GGCATCATCATAGGAGTCACAG-3’ Reverse 5’- AGTCAAAGGGCACCAAGTC-3’	95◦C for 30 s.	61.5◦C for 45 s	72◦C for 1 min
3.	GAPDH forward	Forward 5’- AACGGGAAGCTTGTCATCAATGGAAA-3’ Reverse 5’- GCATCAGCAGAGGGGGCAGAG-3’	95◦C for 30 s.	62◦C for 40 s	72◦C for 1 min

Flow cytometry analysis

The experiment was carried out according to the manufacturer’s instructions (Elabscience, Annexin-V APC apoptosis detection kit: E-CK-A217). T98G and U87 MG glioma cells (2 × 10⁴) were seeded in 60 mm Petri dishes and after 24 h treated with recombinant TRAIL(0.25 µg/mL) and W. somnifera FE 200 µg/mL alone and in combination with TRAIL(0.25 µg/mL) for 24 h. After incubation, all the floated cells and adherent cells (removed using mechanical gentle scrapping^33,34) were collected and centrifuged at 300xg for 5 min. Following this pellets were washed with 1X PBS two times. Next, pellets were then re-suspended into 1x binding buffer, and 5 µl of Annexin APC-PI were added and incubated for 20 min., in the dark at room temperature. Samples were analyzed by using a FACS Caliber flow cytometer (Becton Dickinson). For each sample, 10,000 cell events were collected.

In another set of experiments, T98G glioma cells were treated with USP5-specific siRNA using Interferrin transfection reagent as discussed earlier. After 48 h of siRNA transfection USP5 knockdown cells were treated with TRAIL 0.25 µg/mL and W. somnifera FE 200 µg/mL either alone or in combination for 24 h and subjected to FACS analysis according to manufacturer instructions.

MG132 and chloroquine treatment

T98G and U87 MG glioma cells (2 × 10⁶) were seeded in 60 mm Petri plates. After 24 h of plating, cells were first treated with MG132 and chloroquine 100 μm for 2 h and then with TRAIL 0.25 µg/mL and TRAIL with W. somnifera FE 200 µg/mL for 6 h. Protein lysates were prepared in a cell lysis buffer (Cell signaling) containing protease inhibitors (Abcam) and phosphatase inhibitors (Sigma).

Caspase 2, caspase 9, and caspase 3 colorimetric assay

U87 ​MG and LN229 glioma cells were transfected after 17 ​h of freshly plated cells with specific siRNA’s (CNT (10 ​nM), USP5 (10 ​nM). After 48 ​h of transfection respective treatment with TRAIL ligand (0.25 µg/mL) and W. somnifera FE (200 µg/mL) were given for 24 h. After 72 h of transfection protein lysate was prepared in cell lysis buffer without SDS. Proteins were quantitated with the help of Bradford reagent (BIO-RAD) as per manufacturer protocol. 100​µg of protein was taken for calculating the Caspase 2, Caspase 9, and Caspase 3 cleavage using a caspase colorimetric kit (Biovision) as per the manufacturer’s protocol. Absorbance was taken at 200 or 405 ​nm.

Immunocytochemistry staining

U87 MG and LN229 cells were seeded on a 12 mm coverslip and after 17 h of plating, cells were transfected with siRNA specific for USP5 (10nM) using Interferrin transfection reagent (Polyplus) as per protocol. After 48 h of transfection, cells were treated with either TRAIL 0.25 µg/mL, W. somnifera FE 200 µg/mL, or in a combination of both for 6 h. Remove culture media and gently wash with ice-cooled 1x PBS for 5 min. Fix with 4% paraformaldehyde in PBS for 15 min at room temperature. Permeabilize samples with 5% BSA in PBS for 30 min after washing with 1xPBS. Aspirate blocking buffer and incubated with DR5 primary antibody (Cloud Clone Corp) overnight at 4 degrees. Washed with PBS three times for 5 min and incubated with secondary antibody (Alexa flour 488- Abcam) for 1 h at room temperature. Mounted slides with a drop of mounting media containing DAPI and observed under the fluorescence microscope using 20 x lenses.

High resolution mass spectrometry analysis

Mass spectrometric analyses were performed on a LTQ Orbitrap 6200 series TOF/6500 series Q-TOF B equipped with an ionization mode of ESI source. Parameters were as follow: Collision energy- 0, Fragmentor Voltage-175 V, acquisition method was MS scan. Scan 1 was for full scan (m/z 100–1000) HRMS in positive ionization mode.

cBioportal database and correlation expression

Integrative analysis of Glioblastoma was performed using cBioportal (https://www.cbioportal.org/), a publically available database for tumor genomics and transcriptomic analysis. Using the cBioportal database, we assessed the Glioblastoma (TCGA, Pan Cancer Atlas) study, acquired data of 592 patients for Oncoprint analysis and mRNA correlation analysis of Deubiquitinating enzyme USP5 and Epidermal Growth Factor Receptor (EGFR).

Statistical analysis

The statistical analysis was assessed with the one-way or two-way ANOVA for comparison of data of more than two groups in the study conducted. The student t-test was used for comparison of the two groups only. A two-way contingency table analysis was based on Fisher’s exact test. The p-value > 0.05(*), > 0.01(**), 0.001(***), 0.0001(****) are considered significant.

Results

Cell viability assay on TRAIL and W. somnifera FE treatment in T98G, U87 MG, and LN229 glioma cells

To assess the cell death stimulatory effect: TRAIL ligand and W. somnifera FE on T98G, U87 MG and LN229 glioma cell lines, we administered at two different dosages of TRAIL ligand (0.25 µg/ml, & 1 µg/ml) alone, and three different dosages of W. somnifera FE alone (50 µg/ml, 100 µg/ml, 200 µg/ml) or in combination with TRAIL ligand (0.25 µg/ml) for 48 h. After MTT analysis, we observed that TRAIL ligand alone significantly induced cell death up to 50% with increasing dose in T98G glioma cells. However, in U87 MG and LN229 glioma cells when compared to the control untreated cells there were no cell deaths observed, which signifies that T98G is TRAIL sensitive cell, whereas U87 MG and LN229 as TRAIL resistance glioma cells. In addition, W. somnifera alone at a dose of 50 µg/ml, and 100 µg/ml did not induce cell death, however, 200 µg/ml induced 40% cell death in T98G glioma cells, but at the same concentration, no cell death was observed in U87 MG and LN229 glioma cells (Fig. 1A). Combining TRAIL ligand with W. somnifera FE did not have an additional effect on cell death induced by W. somnifera FE alone in T98G glioma cells, whereas W. somnifera and TRAIL ligand co-treatment did not decrease any cell viability of U87 MG and LN229 cells. This concludes that the cell death seen in combination of TRAIL ligand and W. somnifera FE is due to W. somnifera only, not due to the additional effect of TRAIL ligandin TRAIL sensitive T98G cell line.

Fig. 1.

(A) The cytotoxic effect of TRAIL ligand and W. somnifera FE on TRAIL ligand sensitive (T98G) and TRAIL ligand resistant (U87 MG and LN229) glioma cells: Cell survival (%) of different glioma cells at different concentrations of TRAIL ligand (0.25 µg/ml, and 1 µg/ml), and W. somnifera FE (50 µg/ml, 100 µg/ml, and 200 µg/ml). Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Annexin V-APC PI based apoptotic assay in T98G glioma cells: T98G glioma cells were treated with TRAIL 0.25 µg/mL, W. somnifera 200 µg/ml and in combination for 24 h, were analyzed for early and late apoptosis by Annexin V-APC Propidium Iodide(staining) after passing the treated cells through a flow cytometer. Graph plots based on percentage cells in each quadrant showing a difference in early and late apoptosis upon different treatments. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Annexin V-APC PI based apoptotic assay in U87 MG glioma cells: U87 MG glioma cells were treated with TRAIL 0.25 µg/mL, W. somnifera 200 µg/ml and in combination for 24 h, were analyzed for early and late apoptosis by Annexin V-APC Propidium Iodide(staining) after passing the treated cells through a flow cytometer. Graph plots based on percentage cells in each quadrant showing a difference in early and late apoptosis upon different treatments. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001.

Annexin V-PI staining for apoptotic assay after TRAIL ligand and W. somnifera treatment in T98G and U87 MG glioma cells

We further extended the performed cell viability assay using a more sensitive stage-specific apoptotic cell death using Annexin V-PI staining in T98G and U87 MG cells. We treated T98G and U87 cells with TRAIL ligand (0.25 µg/ml), W. somnifera FE (200 µg/ml), and TRAIL ligand in combination with W. somnifera FE in their respective dosage for 24 h. In T98G glioma cells, treatment with TRAIL showed 28.22% of cells undergoing late apoptosis and 2% undergoing early apoptosis whereas, W. somnifera FE treatment led to 25.33% late apoptosis and 7% early apoptosis, as here in W. somnifera FE alone noted with more early apoptosis in comparison of TRAIL. Interestingly, a combination of TRAIL and W. somnifera FE treatment showed inhibition of transition from early apoptosis to late apoptosis where cells at late apoptosis state were downregulated to 19.98% compared to the TRAIL alone (28.22%) (Fig. 1B). However, in U87 MG glioma cells showed inhibition of transition from early to late apoptosis in all three different treatments; TRAIL ligand alone, W. somnifera FE alone, and in combination of TRAIL ligand and W. somnifera FE treatment: early (8.18%) to late (3.04%), early (15.05%) to late (3.96%), early (11.08%) to late (3.3%) respectively (Fig. 1C). This concludes that T98G glioma cells are TRAIL sensitive in comparison to the U87 MG glioma cells as shown previously by different authors. But in the case of U87 MG which is TRAIL-resistant and also W. somnifera FE-resistant, non-promoting effects happen in between the early apoptosis and late apoptosis.

Immunoblot analysis of oncoprotein USP5, apoptotic proteins, and autophagy markers after TRAIL ligand and W. somnifera FE treatment in T98G and U87 MG glioma cells

Since W. somnifera and TRAIL ligands could not transit early apoptosis to late apoptosis in U87 MG cells, therefore, we sought to study the signaling intermediates in T98G and U87 MG cells. Previously published reports in T98G glioma cells showed that USP5 upon its cleavage promotes TRAIL ligand-mediated apoptosis. Therefore, we quantitated the expression of USP5 or its cleavage in T98G and U87 MG under two dosages (0.25 µg/ml, and 1 µg/ml) of TRAIL ligand treatment for 6 h. The USP5 protein was cleaved after ligand treatment inT98G glioma cells at a lower dosage(0.25 µg/ml), followed by downregulation of total USP5 protein expression, where cleavage of USP5 remains visible at higher TRAIL dosage(1 µg/ml) as well, whereas, in U87 MG, no such USP5 cleavage or its downregulation was seen (Fig. 2A). Moreover, in another set of experiments, we treated T98G and U87 MG glioma cells with a lower dosage of TRAIL ligand(0.25 µg/ml) compared with W. somnifera FE higher dose was chosen(200 µg/ml) for 6 h treatment, to study the expression of USP5 and other apoptotic proteins indicators. Upon W. somnifera FE treatment in T98G glioma cells, the protein expression of USP5 was seen downregulated whereas USP5 protein expression was upregulated in U87 MG glioma cells (Fig. 2B and C). The various apoptotic and anti-apoptotic proteins including Bcl-2, XIAP, SMAC, and autophagy protein LC3B were estimated. Bcl-2, an anti-apoptotic protein that plays a crucial role in the interplay between autophagy and apoptosis, was downregulated in T98G cells whereas it was upregulated upon W. somnifera FE treatment in U87 MG cells, suggesting a potential switch towards the autophagy pathway on W. somnifera treated U87 MG cells, which was further confirmed by LC3B expression (autophagy marker) analysis which was downregulated in W. somnifera FE treated U87 cells as well as in T98G glioma cells, confirmed no autophagy pathway involved in U87 MG glioma cells and T98G glioma cells. LC3B was downregulated in TRAIL-treated T98G cells, however, LC3B was upregulated in U87 MG TRAIL-treated cells. Additionally, in both the cell lines, SMAC was downregulated upon TRAIL ligand treatment, whereas SMAC was upregulated in U87 MG glioma cells upon W. somnifera FE alone treatment but not in T98G glioma cells. SMAC anti-regulates XIAP, which was also measured and were seen downregulated in T98G cells upon TRAIL & W. somnifera FE alone treatment, whereas no significant change was noted in XIAP protein level in U87 MG cells. This showed aberrant regulation being exchanged between XIAP and SMAC. This suggests SMAC which is an apoptotic protein supposed to be highly expressed in TRAIL-sensitive T98G cells therefore, to understand its aberrant regulation we pretreated the T98G as well as U87 MG cells with MG132 and chloroquine phosphate followed with TRAIL and TRAIL in combination with W. somnifera FE treatment. This experiment showed the SMAC protein expression is higher in T98G cells upon TRAIL treatment after inhibiting the proteasome and lysosome mediated degradation pathway, whereas in U87 MG cells, SMAC expression was not upregulated or marginally accumulated after MG132 or chloroquine treatment. This suggests SMAC expression and its degradation was colloquial with apoptosis in T98G cells, but in U87 MG the pattern of SMAC expression visualized after using a degradation pathway inhibitor was seen differently, making the U87 MG cells more resistant (Fig. 2D and E).

Fig. 2.

(A) T98G and U87 MG glioma cells were treated with TRAIL ligand in two different dosages (0.25 µg/ml, and 1 µg/ml) and USP5 protein expression was analyzed. β-actin was used as a loading control. Densitometry analysis was performed with respect to β-actin. (B) T98G glioma cells were treated with TRAIL ligand (0.25 µg/ml), and W. somnifera FE (200 µg/ml). Different proteins like USP5, Bcl-2, SMAC, LC3B, and XIAP were analyzed. β -actin was used as a loading control. Densitometry analysis was performed with respect to β-actin. (C) U87 MG glioma cells were treated with TRAIL ligand (0.25 µg/ml), and W. somnifera FE (200 µg/ml). Different proteins like USP5, Bcl-2, SMAC, LC3B, and XIAP were analyzed. β -actin was used as a loading control. Densitometry analysis was performed with respect to β-actin. (D) T98G glioma cells were treated with MG132 and chloroquine phosphate 2 h before TRAIL ligand (0.25 µg/ml), and TRAIL ligand + W. somnifera FE co-treatment. SMAC protein expression was analyzed. β -actin was used as a loading control. (E) U87 MG glioma cells were treated with MG132 and chloroquine phosphate 2 h before TRAIL ligand (0.25 µg/ml), and TRAIL ligand + W. somnifera FE co-treatment. SMAC protein expression was analyzed. β -actin was used as a loading control.

Time and dose-dependent study of W. somnifera FE treatment in U87 and T98G glioma cells reveals the upregulation of USP5 protein in TRAIL-resistant U87 glioma cells

Since our previous data showed the upregulation of oncoprotein USP5 in TRAIL-resistant U87 MG glioma cells, therefore we made lysate for analyzing quantitative expression change in USP5 after W. somnifera FE treatment in two different doses (100 µg/ml, 200 µg/ml) for 12 h and 24 h in both the cell lines. Here, we have seen that USP5 was upregulated in 100 µg/ml, and 200 µg/ml after 12 h W. somnifera FE treatment which was further increased after 24 h in the same doses in the U87 MG glioma cell line, whereas no such upregulation was seen in T98G glioma cells after W. somnifera treatment in same doses and same period. SMAC protein expression was also analyzed and showed upregulation in 100 µg/ml, and 200 µg/ml doses of W. somnifera treatment at 24 h, whereas in the case of T98G SMAC was upregulated at 100 µg/ml W. somnifera treatment for 12 h afterward started down-regulated with dose and time-dependent treatment of W. somnifera FE. Along with SMAC, XIAP an inhibitor of apoptosis that works antagonist to SMAC protein was analyzed and showed no change in expression in both the cell lines after W. somnifera (chloroform unsolubilized fraction)treatment in a dose and time-dependent manner. Interestingly, another deubiquitinating family protein TNFAIP3 was downregulated upon W. somnifera treatment in U87 MG glioma cells whereas in case of T98G TNFAIP3 protein was in ubiquitinated form (Fig. 3A and B). This data set confirms the upregulation of oncoprotein USP5 in TRAIL-resistant U87 MG glioma cells after W. somnifera treatment, along with upregulated SMAC expression but failed to induce apoptosis.

Fig. 3.

(A) Western blot analysis of USP5, SMAC, XIAP, and TNFAIP3 in T98G glioma cells after W. somnifera FE (100 µg/ml, and 200 µg/ml) treatment for 12 h and 24 h. Densitometry analysis was performed with respect to β-actin. (B) Western blot analysis of USP5, SMAC, XIAP, and TNFAIP3 in U87 MG glioma cells after W. somnifera FE (100 µg/ml, and 200 µg/ml) treatment for 12 h and 24 h. Densitometry analysis was performed with respect to β-actin. (C) T98G glioma cells were analyzed for DR4 and DR5 expression using semi-quantitative PCR. GAPDH was used as a loading control. (D) U87 MG glioma cells were analyzed for DR4 and DR5 expression using semi-quantitative PCR. GAPDH was used as a loading control.

Semi-quantitative PCR analysis of DR4 and DR5 TRAIL receptors in T98G and U87 MG glioma cells after TRAIL ligand and W. somnifera FE treatment

We further investigated the expression of DR4 and DR5 TRAIL receptors involved in inducing apoptosis in TRAIL-sensitive T98G cells and TRAIL-resistant U87 MG cells. In this experiment, we analyzed transcript expression of DR4 and DR5 TRAIL receptors by semi-quantitative PCR methodology, at three different cDNA concentrations (25ng, 50ng, 100ng) taken after different treatments: TRAIL ligand alone(0.25 µg/ml), W. somnifera FE alone(200 µg/ml), and in cotreated TRAIL ligand and W. somnifera FE for 6 h. From the treated experimental samples mRNA was isolated and transcribed cDNA was used to demonstrate the study. In T98G glioma cells, we observed that the expression of DR4 was upregulated in the presence of TRAIL alone (0.25 µg/ml) treatment. Similar upregulation of DR4 expression was also observed with W. somnifera FE (200 µg/ml) alone treatment, whereas when W. somnifera FE was combined with TRAIL showed downregulation of DR4 expression (Fig. 3C). Additionally, another TRAIL receptor DR5 was also analyzed, which showed its upregulation in TRAIL treatment alone, but was downregulated in W. somnifera alone or W. somnifera in combination with TRAIL ligand. This suggests T98G glioma cells are sensitive towards TRAIL ligand stimulation which was confirmed through upregulation of DR4 and DR5 TRAIL receptors but at the same time in W. somnifera FE treated and TRAIL ligand co-treated samples showed downregulation of DR5 & DR4. On the other hand, although the expression of DR4 and DR5 is comparatively less in U87 MG, where TRAIL ligand alone treatment itself is not enough to increase the expression of either DR4 or DR5 TRAIL receptor, the same has been observed in W. somnifera FE treated or in TRAIL ligand co-treated with W. somnifera FE (Fig. 3D). Therefore, in concordant with the previous analysis, the transition inhibition from early to late apoptosis in TRAIL and W. somnifera FE co-treated T98G and U87 MG cells is due to the downregulation of TRAIL receptors DR4 or DR5 also along with USP5 upregulation in U87 MG cell line.

Annexin V-PI staining in SiRNA mediated knockdown of USP5 in T98G, U87 MG, and LN229 glioma cells treated with TRAIL ligand, W. somnifera FE

Since U87 MG cells have higher USP5 expression after W. somnifera treatment whereas in T98G cells W. somnifera treatment showed no effect on USP5 expression with significant late apoptosis, USP5 overexpression could be the reason behind the inhibition of early to late apoptosis transition in TRAIL-resistant glioma. We further insight into the role of USP5 in glioma cells, and conducted USP5 knockdown experiments in both T98G and U87 MG cells. Interestingly, in T98G glioma, we observed that knockdown of USP5 showed early apoptosis (0.22%), late apoptosis (12.41%), and necrosis (28.76%), followed by treatment with W. somnifera FE for 24 h, this did not significantly induce cell death and showed early apoptosis(1.12%), late apoptosis(15.2%), and necrosis(13.9%), whereas the knockdown of USP5 in T98G cells further treatment with TRAIL ligand for 24 h resulted in early apoptosis(0.74%), late apoptosis(93.15%), and necrosis(4.86%)Fig. 4A(I). The knockdown of USP5 was confirmed through western blotting. Notably, the expression of LC3II, a marker for autophagosome formation, was highly elevated in only USP5 knockdown T98G cells treated with W. somnifera FE (chloroform unsolubilized fraction), suggesting that after knockdown of USP5 followed by W. somnifera FE treatment, autophagy cell death occurs instead of apoptosis (Fig. 4A(II)). We have performed the immunofluorescence staining of TRAIL receptor (DR5) in T98G cells after TRAIL ligand (0.25 µg/ml) treatment showed upregulated DR5 expression confirms the activation of apoptosis through death receptor 5 (DR5) of TRAIL ligand (Fig. 4A(III)). This data set concordant with Fig. 3A concludes that USP5 in T98G after TRAIL treatment gets cleaved which is bifunctional, so it suppresses TRAIL-mediated sensitization in T98G cells. The knockdown of USP5 completely sensitizes T98G cells to TRAIL-mediated apoptosis.

Fig. 4.

(A) (I) Knockdown of USP5 and control with specific siRNAs in T98G glioma cells were performed. After 48 h of siRNA transfection, cells were treated with TRAIL 0.25 µg/ml and W. somnifera FE alone and in combination for 24 h, results were comparatively analyzed, and the early and late apoptosis quadrant by Annexin V APC-Propidium Iodide staining were showed using flow cytometry. Graph plots based on percentage cells in each quadrant show the difference in early and late apoptosis upon different treatments. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (II) Western blot data showing USP5 expression along with LC3 expression after knockdown of USP5 followed by TRAIL and W. somnifera FE treatment. β -actin was used as the loading control. Densitometry analysis was performed with respect to β-actin. (III) Immunofluorescence staining of DR5 after TRAIL ligand(0.25 µg/ml) treatment in T98G glioma cells, DAPI blue colour stain is nucleus . Graph showing the fluorescence intensity in respective treatment. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (B) (I) Knockdown of USP5 and control with specific siRNAs in U87 MG glioma cells were performed. After 48 h of siRNA transfection, cells were treated with TRAIL 0.25 µg/ml and W. somnifera FE alone and in combination for 24 h, results were comparatively analyzed, and the early and late apoptosis quadrant by Annexin V APC-Propidium Iodide staining were showed using flow cytometry. Graph plots based on percentage cells in each quadrant show the difference in early and late apoptosis upon different treatments. Graph plots based on percentage cells in each quadrant show the difference in early and late apoptosis upon different treatments. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (II) Western blot data showing USP5 expression after knockdown of USP5 followed by TRAIL and W. somnifera FE treatment. β -actin was used as the loading control. Densitometry analysis was performed with respect to β-actin. (III) Caspase 2, Caspase 9, and Caspase 3 colorimetry assay performed in U87 MG after W. somnifera FE treatment (200 µg/ml), and USP5 knockdown followed by W. somnifera FE (200 µg/ml), and TRAIL ligand (0.25 µg/ml). Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (IV) Western blotting of USP5, SMAC in U87 MG cells in the same lysate that is used for Caspase assay. Densitometry analysis was performed with respect to β-actin. (C) (I) Knockdown of USP5 and control with specific siRNAs in LN229 glioma cells were performed. After 48 h of siRNA transfection, cells were treated with TRAIL 0.25 µg/ml and W. somnifera FE alone and in combination for 24 h, results were comparatively analyzed, and the early and late apoptosis quadrant by Annexin V APC-Propidium Iodide staining were showed using flow cytometry. Graph plots based on percentage cells in each quadrant show the difference in early and late apoptosis upon different treatments. Graph plots based on percentage cells in each quadrant show the difference in early and late apoptosis upon different treatments. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (II) Western blot data showing USP5 expression after knockdown of USP5 followed by TRAIL and W. somnifera FE treatment. β-actin was used as the loading control. Densitometry analysis was performed with respect to β-actin. (III) Caspase 2, Caspase 9, and Caspase 3 colorimetry assay performed in LN229 after W. somnifera FE treatment (200 µg/ml), and USP5 knockdown followed by W. somnifera FE (200 µg/ml), and TRAIL ligand (0.25 µg/ml). Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (IV) Western blotting of USP5, SMAC in LN229 cells in the same lysate which is used for Caspase assay. β-actin was used as the loading control. Densitometry analysis was performed with respect to β-actin.

In contrast, in U87 MG cells, we previously observed that the apoptosis is inhibited in the early to late transition stage which after USP5 knockdown led to a significant increase in apoptosis when treated with W. somnifera FE (chloroform unsolubilized fraction). The late apoptosis was 50.85%, with only 0.45% early apoptosis, compared to USP5 knockdown cells without W. somnifera treatment, which had a late apoptosis rate of only 6.17% and 0.94% early apoptosis. When USP5 knockdown cells were treated with TRAIL ligand, late apoptosis (6.05%) and early apoptosis (1.23%) were observed Fig. 4B(I)). The knockdown of USP5 was confirmed through western blotting (Fig. 4B(II)). Along with FACS analysis, to confirm the apoptosis pathway induced upon W. somnifera FE treatment after si USP5 we did the caspase 2, caspase 9, and caspase 3 colorimetric assay. The caspase 2 assay showed low activation of caspase 2, whereas high activity of caspase 9 and caspase 3 in si USP5 with W. somnifera FE cells confirms the involvement of intrinsic pathway of apoptosis in the following treatment (Fig. 4B(III)). The same lysates were run for western blotting analysis showed significant USP5 knockdown and SMAC was also upregulated in si USP5 W. somnifera FE treated sample which confirms the induction of apoptosis after USP5 knockdown upon W. somnifera treatment (Fig. 4B(IV)). This set of data concludes that USP5 in TRAIL resistance U87 MG glioma is inhibiting early to late apoptotic transition confirmed after knockdown of USP5 which results in late apoptosis in U87 MG cells.

To support the U87 MG data we have used LN229, another glioma cell line that reportedly is TRAIL-resistant. Like U87, LN229 also showed inhibition of apoptosis in the early to late transition stage (18.4–1.5%) in W. somnifera FE treatment and necrosis 0.5% was noted. LN229 also showed upregulation of USP5 and SMAC upon W. somnifera FE treatment in a time and dose-dependent manner. Along with this, downregulation of TNFAIP3 protein was seen, ubiquitinated form of TNFAIP3 was also observed and showed no effect due to W. somnifera FE treatment. (Fig. 4C(II)). Since there was higher USP5 expression, this led us to knockdown USP5 in LN229 glioma cells showed early apoptosis 0.8%, late apoptosis 1.05%, and necrosis 2.1%. After knockdown of USP5 with W. somnifera FE treatment for 24 h converted early apoptotic 0.67% into late apoptosis 58.37% significantly and necrosis 15.35%, whereas USP5 knockdown with TRAIL ligand treatment did not show such effect as early apoptosis was 3.35%, late apoptosis 0.84%, and necrosis 0.5% (Fig. 4C(I)). The Caspase 2, Caspase 9, Caspase 3 colorimetric assay, and western blot analysis of the LN229 cell line were in concordance with U87 MG cells (Fig. 4C (III and IV)). Furthermore, SMAC upregulation in W. somnifera FE without change in XIAP, was extended with experiment in relation to the USP5 protein upregulation in W. somnifera treated cells. In Fig. 4B(IV) and 4C(IV) the comparative SMAC expression was showed relatively much higher only in USP5 siRNA + W. somnifera FE in comparison to W. somnifera FE treated only. This significant upregulated expression of SMAC was also found correlated with Caspase 9 and Caspase 3 activation. Therefore, USP5 dependent SMAC downregulation promotes cell survival upon W. somnifera treatment. Here, it confirmed that W. somnifera FE in TRAIL resistance glioma cells could induce apoptosis in the absence of USP5 which inhibits the early to late apoptosis transition in TRAIL-resistant U87 MG and LN229 glioma cells.

Immunofluorescence study of DR5 expression in U87 MG and LN229 glioma cells after USP5 knockdown, treated with TRAIL ligand and W. somnifera FE (chloroform unsolubilized fraction)

Since U87 MG and LN229 glioma cells showed apoptosis only in USP5 knockdown followed by treatment with W. somnifera FE extract; we further studied the downstream signaling cascade for the apoptotic induction. Western blot analysis revealed that W. somnifera FE treatment after USP5 knockdown upregulates the DR5 expression significantly in U87 MG and LN229 glioma cells. The expression of DR5 was downregulated in TRAIL ligand, as well as in USP5 knockdown co-treated with TRAIL ligand in both the TRAIL resistant glioma cell lines, this may be the reason for not inducing the apoptotic signaling cascade in respective treatments Fig. 5A(I) and 5B(I). Along with DR5, we have also analyzed TNFAIP3 protein expression that showed treatment of W. somnifera inhibited the ubiquitinated form of TNFAIP3 irrespective of presence of absence of USP5, whereas, TNFAIP3 protein was highly upregulated in USP5 knockdown co-treated W. somnifera U87 MG cells. TNFAIP3 gets ubiquitinated in USP5 knockdown cells and USP5 knockdown cells co-treated with TRAIL ligand which signifies that aside of DR5 expression TNFAIP3 is important for inducing apoptosis in the absence of USP5. In our previous studies we reported that for inducing apoptosis co-knockdown of USP5 and USP8 is necessary. So we analyzed USP8 protein in USP5 knockdown U87 cells which showed stable expression of USP8 after USP5 knockdown as well as in USP5 knockdown with W. somnifera treatment. USP8 gets downregulated after TRAIL treatment in USP5 knockdown U87 MG cells, still this did not induce apoptosis, which signifies the presence of any other protein that is highly expressed and inhibiting the apoptosis even in absence of both USP5 as well as USP8 after TRAIL treatment (Fig. 5A(I)). Furthermore, immunofluorescence data revealed that W. somnifera FE treatment after the knockdown of USP5 showed a higher relative fluorescence intensity of DR5 was 4.17 in U87 MG cells and 4.8 in LN229 glioma cells compared to the relative fluorescence intensity of control 2.20 and 2.27 in U87 MG and LN229 glioma cells respectively (Fig. 5A(II) and 5B(II). In Fig. 5A(III) and Fig. 5B(III), the graph represents the relative fluorescence intensity of DR5 in U87 MG and LN229 respectively after treatment with W. somnifera (200 µg/ml), knockdown of USP5 using specific siRNA, and si USP5 + W. somnifera (200 µg/ml), si USP5 + TRAIL ligand (0.25 µg/ml), TRAIL ligand (0.25 µg/ml) showing highest fluorescence intensity in W. somnifera FE + si USP5 cells.

Fig. 5.

(A) U87 MG glioma cells: Immunofluorescence staining (Methodology section) and western blotting of DR5 in U87 MG cells after W. somnifera FE (200 µg/ml), USP5 knockdown using specific si RNA, USP5 knockdown followed by W. somnifera FE treatment (200 µg/ml), and TRAIL ligand treatment(0.25 µg/ml), and TRAIL ligand alone treatment (0.25 µg/ml): (I) Western blotting analysis showing DR5 expression in respective treatments. Densitometry analysis was performed with respect to β-actin. (II) Immunofluorescence staining of DR5 (green colour), blue colour is DAPI (nuclear stain) in respective treatment. (III) Graph showing the fluorescence intensity in respective treatment. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (B) LN229 glioma cells: Immunofluorescence staining and western blotting of DR5 (green colour) and DAPI blue colour stain is nuclear in LN229 cells after W. somnifera FE (200 µg/ml), USP5 knockdown using specific si RNA, USP5 knockdown followed by W. somnifera FE treatment(200 µg/ml), and TRAIL ligand treatment(0.25 µg/ml), and TRAIL ligand alone treatment (0.25 µg/ml): (I) Western blotting analysis showing DR5 expression in respective treatments. Densitometry analysis was performed with respect to β-actin. (II) Immunofluorescence staining of DR5 Green colour & nucleus is in blue colour (DAPI) in respective treatment. (III) Graph showing the fluorescence intensity in respective treatment. Data are expressed as the mean ± SE. The * mark represents significant results, *p < 0.05, **p < 0.01, ***p < 0.001. (C) High-resolution Mass spectrometric spectra of different compounds present in W. somnifera FE (chloroform unsolubilized fraction). (D) The independent expression of USP5 and EGFR was projected using Oncoprint data analysis. The data collected from Glioblastoma (TCGA, Pan Cancer Atlas) dataset accessed through cBioportal. Sign of different genetic alternation proposed towards USP5 and EGFR in individual patient glioma tumor was represented in the figure. Green highlighted patient samples were showing missense mutation in EGFR and USP5, whereas red highlighted patient samples were showing EGFR and USP5 gene amplified. A two-way contingency table showing EFGR wild type and amplified and USP5 wild type and amplified are not mutually exclusive. Statistical analysis was performed using Fisher’s exact test. (E) mRNA correlation of EGFR and USP5 gene was assessed using cBioportal. The Glioblastoma (TCGA, Pan Cancer Atlas) analyzed dataset: Y-axis represents mRNA expression of EGFR gene and X-axis represents mRNA expression of USP5 gene. The negative correlation was projected during the analysis, Spearmen co-efficient (-0.06) and Pearson co-efficient (-0.04) and p-value = 0.616.

Abundance of characterized compound in experimental treatment segment visualized by high resolution-mass spectrophotometer

In the present study, an HRMS/MS method for profiling and characterization of constituents present in the chloroform pretreated non-soluble extract was dissolved in 100% methanol. This W. somnifera FE extraction was undergone following HRMS/MS based characterization and analysis. We have characterized most of the compounds based on the study published by other group³⁵ (Supplementary Fig. 2). In our study HRMS/MS conditions were optimized to obtain the maximal chromatographic resolution and MS signal using ESI ionization mode shows a representative chromatogram of W. somnifera FE under optimal conditions. Based on the previously reported studies in W. somnifera fruit crude extract, our chloroform pretreated non-soluble extract (Fig. 5C), chromatogram analysis showed following enriched enlisted compounds- Withagenin A Diglucoside(abundance-12829.46), Withanoside IX(abundance-1868.22), Sominone Triglucoside(abundance-1868.22), Withanoside III(abundance-12829.46, 12537.21), Withaferin A (abundance-8505.81,11576.81), 23,24-dihydrowithagenin, Daturmalakoside(abundance-1726.39, 2518.96), Withanoside II(abundance-2518.96), Dehydrowithanoside III(abundance-1875.21), Vicosalactone B(abundance-4072.28), Withanoside I(abundance-2518.96), 23, 24 Dihdrwithaferin A(abundance-11576.21), Withaferin A isomer (abundance-11576.81), Withanamide J, K, M and Q(abundance-3230.25, 3124.17, 8505, 14740.93), Withanamide J isomer(abundance-14740.93), Methoxyferuloyltyramine, Feruloyltyramine(abundance-3701), Hydroxypalmitic acid diglucoside(2335.45, 2908.94), Grossamide (2905.39, 9829). Few more compounds with high abundance were showed with m/z peak of 149.0246 and 279.1614 in our extract were not identified based on the literature search and have an abundance of 32194.37, and 20229.89 (Supplementary Fig. 3). From our experimental studies, the used enlisted compounds present in W. somnifera FE isolated extract were proposed in cancer cell promotion.

The correlation between EGFR and USP5 across the existing database of glioma tumor sample

Additionally, we sought to understand the relationship between the deubiquitinating enzyme USP5 and Epidermal Growth Factor Receptor (EGFR) at its mRNA level. The dataset for 592 glioma tumor patients was acquired and analyzed using cBioportal data repository unit. These access data for USP5 and EGFR was analyzed in each patient tumor. This showed the mutation rate of USP5 gene in access data is only 3% altered and EGFR gene is 47% altered. We further checked the mutual exclusivity of two genes where non mutated/amplified cases of USP5 and EGFR was analyzed. The analysis showed 304 (51.35%) patient cases with both wild type EGFR mRNA and USP5 mRNA, whereas 270 (45.6%) patients with amplified EGFR mRNA in wild type USP5 mRNA. Interestingly, their was only 13 (2.19%) patients with amplified USP5 mRNA in wild type EGFR mRNA cases and only 5 (0.8%) patients showed both USP5 and EGFR mRNA amplified. The p-value for the mutual exclusivity of EGFR mRNA and USP5 mRNA was 0.13 based on Fisher’s exact test which is not significant (Fig. 5D). This concludes that EGFR overexpression in more prevalent in GBM tumor patient where USP5 is not found correlated or any such mutual exclusive event exist. Along with this, we have performed mRNA correlation analysis, suggested a negative correlation in between EGFR and USP5 gene is also not significant (p-value = 0.4), Spearmen co-efficient (-0.06), and Pearson co-efficient (-0.04) (Fig. 5E). Therefore, elevation of USP5 expression in W. somnifera FE treatment is an independent event of EGFR.

Discussion

Withania somnifera is a traditional herb used to treat various diseases in developing countries. Mainly to date, Withania somnifera leaves, and root extracts were characterized as carrying anti-cancerous properties³⁶. It has emerged as a potent candidate in cancer chemoprevention due to its ability to intervene in several biological pathways, including apoptosis, angiogenesis inhibition, stress response modulation, and immune regulation. The capacity of WS and its bioactive components, particularly withaferin A (WA) is to induce apoptosis in cancer cells³⁷. The induction of programmed cell death is central to cancer chemoprevention, where clearing impaired cells can halt tumor development. It has been reported that the component of Withania somnifera fruit includes secondary metabolites like alkaloids, tannins, reducing sugar, saponins, terpenoids, coumarin, oil, and fats³⁸ These components are known to have antibacterial activity and anti-oxidant activity and thus, can be used against neurodegenerative diseases like Parkinson’s disease and Alzheimer’s disease³⁹. People in developing countries mostly in Africa and Asia choose to take the benefit of traditional herbs such as W. somnifera in treating various diseases but due to lack of cancer diagnosis and appropriate therapy end up to the late stage cancer⁴⁰.

Isolation or extraction of compounds from different parts of the plant being carried out based on ecological backgrounds shows the variation in abundance of phytochemicals obtained from different parts. Underlying studies suggested that the different extraction procedures may significantly fluctuate the phytochemical content and its combination. Furthermore, different solvent pre-treatment while extracting the metabolites may influence the phytochemical yield and therefore its respective anti-cancerous characteristics. In our study, the chloroform pre-treatment was used to remove to majority of fatty acids, and alkaloids before the preparation of the chloroform non soluble methanolic extract.

In present study, the potential of W. somnifera FE extract were shown not only as an anti-cancerous agent but also has a cancer promoting property by uniquely promoting cell survival via elevating the deubiquitinating enzyme protein USP5. The current study explores the effects of Withania somnifera fruit extract (FE) on TRAIL ligand-sensitive T98G and TRAIL ligand-resistant U87 MG & LN229 glioma cell lines, with a particular focus on the regulatory role of a particular solvent preparatory strategy utilized for preparation of W. somnifera extract. The mechanism behind resistance against W. somnifera FE lies in its solvent preparatory strategy because of removal of a few of the Withanolides like Withanoside IV after chloroform pre-incubation (chloroform solubilized), were confirmed of its presence after running through the HPLC C18 column, where mobile phase was comprising of 50% acetonitrile and 50% methanol. Compound presence showed at the retention time of 19.865(Supplementary Fig. 4). Therefore, absence of Withanolide compounds at its particular composition, glioma cells may be more resistant to apoptosis upon treatment, this suggests many compounds in particular composition are cancer promoting. Since, W. somnifera, so far carrying the well-established anti-cancerous effect but after further categorizing the W. somnifera fruit constituents on the basis of our pre chloroform treatment strategy led us to identify the constituents which were actually not only alleviating the cancer, but are cancer promoting as well. The deubiquitinating enzyme family protein USP5 was consistently shown as an important oncoprotein expresses during W. somnifera FE treatment in U87 and LN229 as well as in T98G, where W. somnifera FE mediated anti-cancerous effect is prominent only in absence of USP5 enzyme, otherwise is inducing cell survival or apoptosis limited only to early stage. In support we published previously that the knockdown of USP5 is not sufficient to introduce apoptosis, additionally where USP8, an another deubiquitinating enzyme has to be depleted to induce apoptosis in glioma cells⁴¹. Recently, the function of USP5 potentially plays an important role in modulating immunotherapy by regulating the deubiquitination of PD-1(programmed cell death − 1 protein), and tumor microenvironment to build a tumor rejection⁴². Interestingly, moreover the effect of proposed W. somnifera extract wasn’t inducing a apoptosis programming but was particularly enhanced upon knockdown of USP5 in U87 MG and LN229.

To finally conclude about solvent preparatory strategies, we have performed the comparative cell viability assay in U87 and LN229 glioma cells using the chloroform solubilized obtained filtrate and the chloroform non-solubilized fraction dissolved in methanol. Cell viability assay showed that chloroform solubilized W. somnifera FE extract showed cell death at 40 µg/ml in U87 MG and LN229 glioma cells (Supplementary Fig. 5). This confirms that chloroform pretreatment removed the compounds that have anti-cancerous or anti-cell viability properties. This investigation while dealing with the whole crude form of W. somnifera fruit extract across the diversified population living in under-developing countries where cancer awareness among the people is lacking⁴³. Since, the origination of glioma is still obscure- CMV virus impacts patient survival by tumor progression or environmental pollutants like PFAS found in higher concentration in glioma male patients and regulates cell proliferation through Ki-67, and p53 pathways and recently the model of glioma genesis been postulated on taking the traumatic brain injury site where the non-mutated cells were of neural crest like cells (NCC) and mesenchymal stem cells (MSC) type as part of regenerative changes, suggested the activation of early injury like alteration during the early tumorigenesis^44–46. On the other side, Withania somnifera extract resulted in increase in neuron projection and less neuronal cell death in traumatic brain injury (TBI) model and inhibited cell proliferation of HCV virus through downregulation of TNF-α^47,48. Such studies were carrying insightful information to build the Withania somnifera mediated effect to lead as anti-cancerous or neuroprotection where early tumor stage mimics with regenerative response in brain injury site. Therefore, categorizing the constituent component of W. somnifera fruit extract would plays an essential step in upregulating the USP5 and downregulation of DR5 drive events are in case would be non-anti-cancerous or neuroprotection or could be cancer promoting. The constituent composition may be play a unique role in therapeutic benefit.

Electronic supplementary material

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Abbreviations

DR5

Death receptor 5

EGFR

Epidermal growth factor receptor

SMAC

Second- MITOCHONDRIA DERIVED ACTIVATOR OF CASPASE

TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand

USP5

Ubiquitin specific peptidase 5

W. somnifera FE

Withania somnifera fruit extract

Author contributions

Ajay Kumar Yadav: Conceptualization, Methodology, Validation, Resources, Writing-Original Draft, Visualization, Supervision, Project administration, Funding acquisition. Sachin Bhardwaj: Write up, Investigation, Experimental set up and designing, Formal analysis, Visualization.

Funding

Financial support from Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Maintenance Grant and Institute of Eminence(IoE) Grant, 2021–2023, University of Delhi and CSIR SRF (09/045(1769)/2019-EMR-I).

Data availability

All Data generated or analysed during the current study are included in the published article in its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participation

Protocols for handling human cells were approved by the Ethics Committee of Dr. B. R Ambedkar Center for Biomedical Research (No.F.50 − 2/Eth.Com/ACBR/16/2379).