Activation of lysosomal mediated cell death in the course of autophagy by mTORC1 inhibitor

Lysosomal biogenesis plays a vital role in cell fate. Under certain conditions, excessive lysosomal biogenesis leads to susceptibility for lysosomal membrane permeabilization resulting in various pathological conditions including cell death. In cancer cells apoptosis machinery becomes dysregulated during the course of treatment, thus allows cancer cells to escape apoptosis. So it is therefore imperative to identify cytotoxic agents that exploit non-apoptotic mechanisms of cell death. Our study showed that pancreatic cancer cells treated with SDS-203 triggered an incomplete autophagic response and a nuclear translocation of transcriptional factor TFEB. This resulted in abundant biosynthesis and accumulation of autophagosomes and lysosomes into the cells leading to their death. It was observed that the silencing of autophagy genes didn’t alter the cell fate, whereas siRNA-mediated silencing of TFEB subdued SDS-203 mediated lysosomal biogenesis and associated cell death. Further mouse tumors treated with SDS-203 showed a significant reduction in tumor burden and increased expression of lysosomal markers. Taken together this study demonstrates that SDS-203 treatment triggers non-apoptotic cell death in pancreatic cancer cells through a mechanism of lysosome over accumulation.


Lysosomal biogenesis plays a vital role in cell fate. Under certain conditions, excessive lysosomal biogenesis leads to susceptibility for lysosomal membrane permeabilization resulting in various pathological conditions including cell death. In cancer cells apoptosis machinery becomes dysregulated during the course of treatment, thus allows cancer cells to escape apoptosis. So it is therefore imperative to identify cytotoxic agents that exploit non-apoptotic mechanisms of cell death. Our study showed that pancreatic cancer cells treated with SDS-203 triggered an incomplete autophagic response and a nuclear translocation of transcriptional factor TFEB. This resulted in abundant biosynthesis and accumulation of autophagosomes and lysosomes into the cells leading to their death. It was observed that the silencing of autophagy genes didn't alter the cell fate, whereas siRNA-mediated silencing of TFEB subdued SDS-203 mediated lysosomal biogenesis and associated cell death. Further mouse tumors treated with SDS-203 showed a significant reduction in tumor burden and increased expression of lysosomal markers. Taken together this study demonstrates that SDS-203 treatment triggers non-apoptotic cell death in pancreatic cancer cells through a mechanism of lysosome over accumulation.
Autophagy is the conserved catabolic cellular process in which lysosomal enzymes degrade defective or excess organelles and unwanted proteins 1 . It is also important for the recycling of metabolites for cell survival, thereby playing a definite role in maintaining cellular homeostasis 2,3 . Autophagy is activated under various stress conditions such as amino acid starvation, unfolded protein response and is inhibited by the high energy state of the cell through activation of mammalian target of rapamycin (mTORC1) 4,5 . mTORC1 is a serine/threonine kinase that regulates cellular metabolism and promotes cell growth 6,7 . mTORC1 activation inhibits mammalian autophagy directly by inhibiting ULK1 complex formation and at transcriptional level it regulates autophagy by modulating the subcellular localization of transcription factor EB (TFEB) 8,9,10 . The deregulation of autophagy pathway compromises cell survival and its association has been found to play a role in many diseases like cancer 11 . Autophagy-lysosome pathway is associated with various cancer hallmarks like death resistance, escaping immune surveillance and deregulation of metabolism 12 . It is established that some invasive cancers s like pancreatic adenocarcinoma are resistant to Type I (apoptotic) and Type II (autophagic) cell death [13][14][15][16][17] ; wherein the activation of nonclassical death pathways through small molecules offers alternate means to address these challenges. Pancreatic cancer is one of the most aggressive human malignancies and is the fourth major cause of cancerrelated deaths 18,19 . Generally, the survival rate of patients with pancreatic adenocarcinoma is very low because of the late diagnosis and poor response to available treatments 20 . Moreover, pancreatic tumor dormancy, relapse along with chemo-resistance liked with autophagy is a major concern for the current available therapies [21][22][23][24] . Studies have shown that some anti-cancerous agents trigger protective autophagy in pancreatic cancer cells and rescue them from apoptosis 25 . Therefore, identification of alternate target-based therapies to circumvent this challenge is a huge unmet medical need. In this study, we identified a novel mTORC1 inhibitor SDS-203 causing non-apoptotic cells death in aggressive MIA PaCa-2 cells. It was observed that SDS-203 triggered early autophagy

SDS-203 induced no-apoptotic death in pancreatic cancer cells.
In our earlier report, we showed that SDS-203 targets mTORC1 and inhibits the cell growth of MIA PaCa-2 cells with IC50 7 ± 1.6 µM 26 . In our current studies, we further tried to understand the mechanism of SDS-203 mediated cell death in MIA PaCA-2 cells, which happened to be non-apoptotic in nature. The initial attempts to characterize the nature of SDS-203 induced cell death via apoptotic pathway were analyzed by flowcytometry and western blotting. Flowcytomtric analysis of Annexin V and propidium iodide (PI) stained MIA PaCa-2 cells revealed that SDS-203 caused non-apoptotic cell death and a large number of treated cells were found in PI + quadrant depicting necrosis ( Fig. 1A-D). This was further confirmed as SDS-203 treatment failed to induce PARP1 or caspase 3 cleavage (Fig. 1E). Free radical generation like reactive oxygen species (ROS) was observed through flowcytometry by using DCFDA that remained unchanged during the course of SDS-203 treatment in MIA PaCa-2 cells ( Supplementary Fig. 1a, b).

SDS-203 treatment induced initial autophagic response in MIA PaCa-2 cells.
To observe the effect of SDS-203 treatment on the autophagic response in MIA PaCa-2, various experiments were performed. SDS-203 treated cells were stained with fluorescent Acridine Orange (AO) dye (1 μg/mL), and cells were observed under fluorescence microscopy. It was found that SDS-203 significantly increased AO acid vesicles in MIA PaCa-2 cells compared to that of control ( Supplementary Fig. 2a, b). Further, we transiently transfected MIA PaCa-2 cells with fluorescent autophagosomal markers GFP-LC3 and followed by treatment with SDS-203 (20 μM). Microscopic analysis clearly showed that SDS-203 increased GFP-LC3 punctation by five fold, when compared to control ( Fig. 2A, B). Treatment of SDS-203 upregulates the conversion of LC3-1 to LC3-II as shown by western blotting (Fig. 2C-D) reflecting the initiation of autophagy process. Expression of other important autophagic proteins ATG5, ATG7 were slightly affected, while Bec lin-1 expressi on remained unchanged by SDS-203 treatment as shown in Figure 2G, H. However, the expression of an adapter protein p62/SQSTM1

SDS-203 triggers initiation of autophagy by mTORC1 inhibition.
Our results confirmed that treatment of SDS-203 (20 µM) at various time points inhibited mTORC1 and its downstream substrates activity significantly (Fig. 3A, B). mTORC1 is a nutrient-sensing kinase that regulates autophagy in cells 27 . Further confirmation showed that SDS-203 action was diminished in presence of known mTORC1 activators-insulin-like growth factor1 (IGF-1) 28 and l -leucine 29 when compared to SDS-203 only treated cells, shown in Figure 3C, D. Cell viability assay (MTT, 48 h) of the same samples demonstrates that IGF or l-leucine treated cells rescued cells death in SDS-203 treated cells (Fig. 3E). These results clearly indicate that SDS-203 mediated inhibition of mTORC1 causing initiation of autophagy. Figure 2G the possibility of autophagy flux blockade or fusion defects between autophagosome and lysosomes in MIA PaCa-2 cells upon SDS-203 treatment. We treated cells with SDS-203 in the presence or absence of autophagy inhibitors-bafilomycin A1 or NH 4 Cl. Western blotting analysis indicated that SDS-203 failed to further upregulate LC3-II expression in pancreatic cancer cells treated with bafilomycin A1 or NH 4 Cl when compared to SDS-203 only treated cells for 6, 12 and 24 h (Fig. 4A, B). On the contrary LC3-II and p62 expression was upregulated upon cotreatment of cells with SDS-203 and autophagy activator rapamycin or starvation when compared with control or individual treatments (Fig. 4C, D). Microscopic examination for endogenous LC3-II puncta formation was intensified in the combined presence of SDS-203 and autophagy inducers when compared with individual treatments ( Fig. 4E-G). These results demonstrated that SDS-203 impairs autophagy flux which leads to the accumulation of autophagosomes and lysosomes thus acts as a potent inhibitor of autophagy maturation.

SDS-203 induces lysosomal biogenesis and accumulation in pancreatic cancer cells.
In the present study, we investigated the lysosomal status in the course of SDS-203 treatment in MIA PaCa-2 cells. Immunofluorescence and immunoblot analysis of lysosomes marker protein LAMP1 revealed that SDS-203 treatment significantly upregulates LAMP-1 expression in MIA PaCa-2 cells (Fig. 5A-D). In addition, SDS-203 treatment upregulated the quantity of the lysosomes observed by confocal microscopy (Fig. 5E, F) and flowcytometry ( Supplementary Fig. 3a, b) using lysotracker staining. Further evaluation by electron microscopy clearly

SDS-203 suppresses autophagy at late stage without affecting lysosomal function and is mediated through suppression of mTORC1.
We tried to find out the effect of SDS-203 on the mobilization and consequent activation of mTORC1 on the lysosomal membrane. For that cells were treated with SDS-203 then processed for colocalization (yellow) evaluation of LAMP1 (green) and mTORC1 (red) by using confocal microscopy. Our results revealed that in control or well-fed MIA PaCa-2 cells LAMP1 and mTORC1 shows colocalization (yellow), while SDS-203 or rapamycin-treated samples showed dispersed fluorescent signals of the respective proteins ( Fig. 6A, B). Similar results were confirmed by Co-immunoprecipitation assay (CO-IP) of the SDS-203 treated MIA PaCa-2 cells (Fig. 6C). Further, our results illustrated that SDS-203 treatment failed to overexpress LC3-II and LAMP-1 expression in transiently mTORC1 upregulated pancreatic cancer cells compared to control ( Fig. 6D-G). Therefore, above data demonstrates that SDS-203 induces lysosomal biogenesis and its accumulation via mTORC1 inhibition.

SDS-203 induces lysosomal biogenesis and its enzyme activity through the mTORC1-TFEB pathway.
After confirming that SDS-203 inhibits the localization of mTORC1 on the lysosomal membrane, we further checked its consequence on downstream proteins transcription factor EB (TFEB) related to lysosomal biogenesis, whose subcellular location is controlled by mTORC1 activity 30 . Our immunoblot and immunofluorescence results confirmed that SDS-203 treatment translocates the TFEB into the nucleus of pancreatic cancer cells ( Fig. 7A-C). Moreover, SDS-203 failed to enhance the expression of LAMP1 in TFEB knocked down MIA PaCa-2 cells ( Fig. 7D-G). MTT assay revealed that SDS-203 treated TFEB knockdown MIA PaCa-2 cells showed increased survival potential compared to scrambled siRNA treated cells ( Supplementary Fig. 4a). To further understand whether SDS-203 induced lysosomal biogenesis was independent of autophagy, western analysis of LAMP1 expression in Beclin1, ATG5, and ATG7 transiently knock out MIA PaCa-2 cells treated with SDS-203 was analyzed. (Supplementary Fig. 4b,c) showed that SDS-203 still maintained the lysosomal biogenesis to several folds in genetically manipulated pancreatic cancer cells compared to sc siRNA-treated cells. Together these results illustrate that SDS-203 translocated TFEB from cytosol to nucleus by inhibiting the activity of mTORC1 which in turn upregulates lysosomal biogenesis independent of autophagy. www.nature.com/scientificreports/ Furthermore, we examined the effect of SDS-203 on lysosomal function by checking its effect on cathepsins, important proteases in the lysosome. Fluorescence measurement of Rhodamine 110 (lysosomal enzyme-substrate) analysis demonstrates that SDS-203 upregulated the Rhodamine 110 signal (the activity of lysosomes) to several folds vs. control (Fig. 7H). Western blotting on other hand revealed that expression of enzymes, particularly cathepsin D and cathepsin L was increased in MIA PaCa-2 cells upon SDS-203 treatment (Fig. 7I, J). Using Rhodamine 110, we investigated whether the upregulation of lysosomal enzyme activity by SDS-203 was also mediated by mTORC1 regulation in MIA PaCa-2 cells. Comparative study ( Supplementary Fig. 4d) demonstrated that SDS-203 was unable to upregulate lysosomal enzyme activity in mTORC1 upregulated MIA PaCa-2 cells. Enzyme activation potency was further downregulated by using mTORC1 activators like l-leucine or IGF-1 in presence of SDS-203. Above results demonstrate that SDS-203 induces lysosomal biogenesis by increasing the lysosomal enzyme activity resulting in cancer cell death independent of autophagy.

Addition of late autophagy inhibitors rescued SDS-203 mediated cell death.
In order to establish the fact that SDS-203 induced pancreatic cancer cell death by upregulation of lysosomal biogenesis and increased its enzyme activity. We treated MIA PaCa-2 cells with late (bafilomycin A1 or NH 4 Cl) and early (wortmannin or 3-methyladenine: 3-MA) autophagy inhibitors in the presence or absence of SDS-203 and then the viability of these cells were checked by MTT assay (Supplementary Fig. 5a, c; Supplementary Fig. 5b, d).
The results clearly illustrate that SDS-203 failed to induce death in MIA PaCa-2 in presence of late autophagy inhibitor, while in the presence of early autophagy inhibitor SDS-203 retained its effect in in ducing cell death. The above data demonstrates that SDS-203 causes lysosomal mediated cell death without any prominat role of autophagy.
In vivo anticancer activity of SDS -20 3 against Ehrlich ascites car cinoma. The above in vitro lea d was taken for in vivo validation in tumor bearing mice by observing, tumor size (Fig. 8A) and volume (Fig. 8B) that were drastically reduced upon SDS-203 treatment compared to un-treated or NH 4 Cl only treated groups, without affecting the average weight of mice (Fig. 8C). Later equal quantity of proteins extracted from the tumor samples were resolved through western blotting for LAMP1 and LC3-II expression (Fig. 8D, E). Our results confirmed that LAMP1 and LC3-II expressions were overexpressed in SDS-203 treated tumor samples compared to vehicle control or NH 4 Cl only treated tumor samples. Collectively, our in vivo data demonstrated that SDS-203

SDS-203 affects multiple targets in pancreatic cancer cells.
The pictorial representation (Fig. 9) shows the mechanism of pancreatic cancer cell death induced by SDS-203. Inhibitory effect of SDS-203 on mTORC1 led to the activation of incomplete autophagy and TFEB nuclear translocation, triggering strong lysosomal biogenesis. Accumulation of lysosomes ultimately leads to the culmination of pancreatic cancer cells. The graphical image was drawn by using the software ChemBioDraw Ultra 14.

Discussion
The majority of the current anticancer therapies are positioned to target apoptotic machinery to induce nonpathological cell death in cancer cells 31 . However, the development of resistance during the course of treatment remains a monumental challenge for successful and effective treatment 32 . During treatment, defects in apoptotic cell death occur due to dysregulation at various programmed steps, which may lead to acquired treatment resistance 33 . Hence the identification of small-molecule agents targeting cancer cells through nonapoptotic machinery is desirable to achieve an effective and lasting therapeutic response 34,35 . In this study, we reported that small-molecule SDS-203 induced cell death in most aggressive pancreatic cancer cells through the non-apoptotic route by up-regulating lysosomal biogenesis and its proteasomal enzyme activity. The functional upregulation of lysosome machinery in MIA PaCa-2 cells upon SDS-203 treatment is mediated via inhibition of mTORC1, as reported earlier by our group 26 . mTORC1 negatively regulates lysosomal function by directly phosphorylating TFEB on the lysosomal membrane, thus allowing interaction between TFEB and 14-3-3 keeping TFEB sequestered in the cytoplasm 36,37 . SDS-203 mediated inhibition of mTORC1 led to the disengagement of TFEB from 14-3-3 allowing TFEB to translocate into the nucleus where it causes activation of various genes associated with autophagy and lysosomal biogenesis. We demonstrated that SDS-203 mediated inhibition of mTORC1 also triggered an early autophagy response in cells as observed by the expression of LC3-II and ATG5. Surprisingly our data revealed an increase in the levels of an adapter protein p62 during the course of treatment, thus indicating an incomplete autophagic response caused by SDS-203.
To better understand the role of SDS-203 triggered autophagy in cell death, we attempted to use both early and late autophagy inhibitors during the course of SDS-203 treatment. The use of early autophagy inhibitors 3-MA and wortmannin were unable to rescue the SDS-203 mediated cell death in MIA PaCa-2 cells. However, the addition of late autophagy inhibitors bafilomycin A1 and NH 4 Cl showed considerable protection against the effect To further apprehend the mechanism of SDS-203 mediated cell death, we tried to understand the role of lysosomal machinery that gets activated during the course of treatment. Microscopic examination and immunoblotting experiments for lysosomal marker protein LAMP1 indicated its enhanced expression in cells treated with SDS-203. Correspondingly, flowcytometric analysis using lysotracker red further supported enhanced lysosomal biogenesis in SDS-203 treated cells. Additionally, hydrolytic lysosomal enzyme activity in SDS-203 treated cells was measured by using nonradioactive fluorescent probe Rhodamine 110 clearly showed that enzyme activity was several folds higher as compared to control or NH 4 Cl treated cells, thereby supporting the role of lysosomes in treating cancer cells. Similarly, the expression of lysosomal proteases Cathepsin D and Cathepsin L were upregulated in SDS-203 treated cells that play a vital role in protein degradation and cell death.
TFEB is the major transcriptional regulator of autophagosomal/lysosomal proteins and its translocation into the nucleus is controlled by its phosphorylated state regulated by mTORC1 38 . Our study found that SDS-203 treatment facilitated the nuclear translocation of TFEB via early inhibition of mTORC1. To further understand the role of TFEB mediated lysosomal cell death under SDS-203 treatment, we performed gene knockdown of TFEB by using siRNA, and observed that the expression of LAMP1 was considerably downregulated. We also observed that TFEB knockdown significantly

Conclusion
Our studies have demonstrated that small molecule SDS-203 induced non-apoptotic death in highly aggressive pancreatic cancer cells. Further, mechanistic understanding revealed that SDS-203 triggered initial autophagic response, resulting in excessive lysosomal accumulation with enhanced proteolytic enzymatic activities causing cell death. These studies remarked the prospective use of SDS-203 against cancers with dysfunctional apoptotic machinery and warrants further pre-clinical studies.   Protein isolation, quantification, western blotting and immunoprecipitation. At the end of the designated treatments, cells were lysed by RIPA or M2 lysis buffer and collected for protein estimation, each steps were done at 4 °C. Following that equal amount of protein from each samples were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 162-0177). Later PVDF membrane was blocked with 5% non-fat milk and cut into strips (based on molecular weight; using Bio-Rad protein standard 10-250 kDa) prior to hybridization with target primary antibodies and HRP + tagged secondary antibodies. Western signaling was detected by using chemiluminescence Horseradish Peroxidase (HRP) substrates and the signals were captured on X-ray film. The density of the various bands in the western blot was quantified using ImageJ software.

Chemicals
Acridine orange staining. Acid vesicular organelles (AVO) formation is a characteristic feature of autophagy induction. To detect the formation of AVO, acridine orange was used. Accumulated AO in acid compartments gives bright red fluorescence (exc = 488 nm laser). 2 × 10 4 cells were seeded in 6 well plates and then treated with SDS-203 in a time-dependent manner (0, 6, 12, 24 h). 15 min before termination AO (1 µg/mL) was added, post staining cells were washed with PBS and taken for microscopy. Data were analyzed by using ImageJ software.
Transfection. GFP-LC3 (plasmid), TFEB siRNA and mTORC1 (WT plasmid) were used to transiently transfect pancreatic cancer cells, later SDS-203 was treated to these transfected cells. MIA PaCa-2 cells were seeded in 6 well plates and allowed to grow up to 70% confluency and then incubated with the plasmid along with transfection reagent, (Fugene kit based transfection reagent; Promega FuGENE HD Transfection Reagent #E2311) in an incomplete media for 14 h. Desired concentration of SDS-203 was later treated to the cells and its effect on the expression of the above-mentioned targets was calculated.
Immunofluorescence and confocal microscopy. Expression of various proteins like LC3-II, mTORC1, LAMP1, GFP-LC3 and intensity of lysotracker DND-red or DCFDA dye in pancreatic cancer cells upon SDS-203 treatment were analyzed by using fluorescence microscopy. 0.2 × 10 4 MIA PaCa-2 cells were allowed to grow on coverslips in 6-well plates, after selected treatment of SDS-203 for particular time points were either incubated with dyes for 15 min before termination or fixed with 4% paraformaldehyde (PFA) for 15 min then permeabilized with (0.02% of Triton-X-100 for 5 min) and later blocked by using blocking buffer (3%BSA + 0.01% Triton-X-100 in PBS for 1 h

Measurement of lysosomal enzyme (CTSB-CTSL) activity.
Enzyme activity of lysosome was done by a nonradioactive method by using rhodamine-110 following protocol 36,37 . Rhodamine 110 (R110) is a sensitive and selective probe for assaying proteases in the cell lysate. This dye contains a fluorogenic substrate called Rhodamine. The substrate can be used to measure lysosomal enzyme activity in cell extracts or purified enzyme preparations using a fluorescence microplate reader. Treated cancer cell samples (SDS-203, NH 4 Cl or SDS-203 + NH 4 CL) were lysed by M2 buffer 20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM glycerol phosphate, 1 mM sodium vanadate and protease inhibitor cocktail for protein extraction. Protein estimation was done in order to incubate equal amount of (25 µg) protein from each sample with 50 µM of Rhodamine110, in 100 µL cell-free system buffer (10 mM HEPES-NaOH pH 7.4, 68 mM sucrose 220 mM mannitol, 2 mM NaCl 2.5 mM KH 2 PO 4 , 0.5 mM EGTA, 2 mM MgCl 2 , 5 mM pyruvate, 0.1 mM PMSF and 1 mM dithiothreitol) in 96 well plate for 1 h at 37 °C.
Reading was taken at an exc/emi wavelength = 380/460 nm by using fluorometry (Tecan Spectra Fluor Plus). Activity was represented as a percentage of fluorescence intensity compared with the control group.
In vivo tumor mice model. The animals were housed under standard husbandry conditions: 24 ± 2 °C temperature, 15-20 complete fresh air changes per hour and 50-60% relative humidity as per guide for the care and use of laboratory animals. Animals were fed with a standard pellet diet (M/S Ashirwad Industries, Chandigarh, India) and autoclaved water was given ad libitum. Approval of the Institutional Animal Ethics Committee, CSIR-Indian Institute of Integrative Medicine, Jammu was sought for the study and number of animals used in all the experiments. SDS-203 was taken for in vivo anticancer assessment against murine tumor model and NH 4 Cl was taken as a negative control. Swiss albino mice (18-23 g) under optimum laboratory conditions were injected with Ehrlich Tumor cells (EAC), grafted from 8 to 10 days old ascites tumor-induced Swiss albino mice. On day zero 1 × 10 7 EAC cells were injected intraperitoneally, later tumor-induced animals were categorized randomly into four test groups, with seven animals per group. The first test group was administrated with normal saline (0.9% i.p.) which act as vehicle control. Another group was treated with SDS-203 (25 mg/kg i.p), and the remaining two groups were treated with or without SDS-203 in presence of NH 4 Cl (20 mg/kg i.p). Treatment was followed up to nine consecutive days; tumor assessment was done on day 12. Tumor measurements included size, weight and volume, and were taken from different groups. Some tumor samples from each group were frozen for protein extraction, which was later done by using tissue lysis buffer and homogenizer. All animal experimental procedures were carried out following the ethical guidelines for the use of animals in experiments and were conducted in compliance with the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA) and the ARRIVE guidelines. All experiments were approved by the animal house CSIR IIIM. The use of experimental animals in this study was approved by the Ethics and Institutional Animal Care and Use, Committees of Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine (CSIR-IIIM).
Ethical approval and consent to participate. The use of experimental animals in this study was approved by the Ethics and Institutional Animal Care and Use, Committees of Council of Scientific and Industrial Research-Indian Institute of Integrative Medicine (CSIR-IIIM) following guidelines of the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA). I confirm that all methods were carried out "in accordance with relevant guidelines and regulations. I confirm that study was carried out in compliance with the ARRIVE guidelines.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.