Cancer cells are selectively eliminated through UPR driven apoptosis during 1 treatment with Aquilegia nivalis extracts

: 27 Background 28 Aquilegia nivalis Flax Jackson, also called Aquilegia vulgaris sub sp. nivalis (Bak.) Brühl 29 or columbine, locally known as “Zoe-neel”, is a wild edible plant traditionally used as an 30 anti-inflammatory medicine by the local nomadic tribes inhabiting the Himalayas of 31 Jammu and Kashmir. The plant has been used as herbal medicine since middle ages in 32 treating ailments that include chronic rhinitis and various infectious diseases. The extracts 33 from the plant possess antioxidant properties and have been reported to be 34 hepatoprotective in rats. Our preliminary studies, however pointed to hitherto unexplored 35 anti-apoptotic potential of the plant which lead us to carry the in-depth study using breast 36 cancer cell lines to validate its anti-cancerous properties and explore the affected 37 pathways. for PARP and caspases revealed that the extract selectively induced apoptosis in 53 cancerous cell lines. UPR markers p-Ire1α and Xbp1 splicing were consistently alleviated 54 in a dose dependent manner, the rate of phosphorylation of eIF2a and ATF4 also 55 decreased with increasing concentration of ANME. The RT PCR results of the RIDD 56 marker, Blos1S1 revealed a similar dose dependent association. The methanolic extract 57 was especially chosen for it could be easily internalized by the cells and any resultant 58 potential bioactive compounds could gain access to the cells because of their hydrophobic 59 nature. 60 Our results suggest that ANME causes deactivation of UPR signaling pathway 62 facilitating apoptosis selectively in cancerous cells, paving the way forward for a novel 63 approach in cancer therapeutics. 64 plant and Some studies have suggested the antiproliferative and pro-apoptotic activities of the plant . Our preliminary data indicated that the plant extracts were toxic to cancerous cells, so the current study was carried out to validate the antiproliferative potential of the plant and explore its 87 molecular targets in the cell. The study revealed that the methanolic extract of the plant affected UPR signaling and RIDD pathways.


ABSTRACT: 27
Background 28 Aquilegia nivalis Flax Jackson, also called Aquilegia vulgaris sub sp. nivalis (Bak.) Brühl 29 or columbine, locally known as "Zoe-neel", is a wild edible plant traditionally used as an 30 anti-inflammatory medicine by the local nomadic tribes inhabiting the Himalayas of 31 Jammu and Kashmir. The plant has been used as herbal medicine since middle ages in 32 treating ailments that include chronic rhinitis and various infectious diseases. The extracts 33 from the plant possess antioxidant properties and have been reported to be 34 hepatoprotective in rats. Our preliminary studies, however pointed to hitherto unexplored 35 anti-apoptotic potential of the plant which lead us to carry the in-depth study using breast 36 cancer cell lines to validate its anti-cancerous properties and explore the affected 37 pathways. 38

39
MTT assay was used to draw the dose response curve and evaluate the effect of 40 increasing concentrations of the extract on cell lines to determine the appropriate dosage 41 to be used for further experimentation. DNA fragmentation analysis was followed 42 through gel electrophoresis and DAPI staining was pursued by phase contrast microscopy 43 to study apoptosis. Quantitative PCR was used to study the expression of UPR signaling 44 and RIDD markers at the level of mRNA. Western blot analysis was used in studying the 45 expression of the various markers of the signaling pathways. The cell cycle analysis was 46 carried out using flow cytometry. 47

Introduction 71
The exploration of natural products offers great opportunity to researchers in medical 72 sciences to identify and explore nature friendly therapeutic agents relevant to different 73 diseases. Aquilegia nivalis is a traditional indigenous herb found in higher altitudes of 74 Himalayas 1 . The plant has been traditionally used in treatment of Asthma and as an anti-75 inflammatory agent. It is also used as a remedy for many ailments including chronic 76 rhinitis and various infections 2 . The extracts of the plant [plant extract] have shown 77 hepatoprotective effects in mice. 3 Several studies have shown the anti-oxidant role of the 78 9 Universal Microplate Reader (Bio-Tek Instruments, USA). The reference wavelength 208 was set at 650 nm. The cell viability of untreated cells was considered 100%. 209

Cell cycle analysis by flow cytometry 210
The cells were seeded at 1 × 10 6 cells/well in a 100-mm culture dish and incubated for 24 211 h. The incubated cells were treated with ANME (50 μg/mL for MDAMB231 100 μg/mL 212 for MCF7 and 25 μg/mL for U87MG), with or without 6μM of Tm for 24 h.. Media was 213 collected in 15ml tubes and cells were washed with PBS containing 0.1% EDTA. 214 Washing solution was also collected. PBS-EDTA was added to the plates followed by 215 incubation at 37 °C for 5-10 minutes. Cells were collected, pipetted up and down and 216 collected in same tubes. Tubes were centrifuged at 1000 g for 5 minutes followed by 217 washing in PBS-Serum (1% serum) and centrifuged again at 1000g. Afterwards, cells 218 were resuspended in 0.5 ml PBS. For fixing, 5 ml ethanol was added drop wise while 219 vertexing the cells. Cells were then stored in deep freezer for FACS analysis. For FACS 220 analysis, fixed samples were centrifuged at 1000g for 5 minutes and washed with PBS-221 serum followed by resuspension in Propidium Iodide-RNAse solution (50 μg/ml 222 propidium iodide, 10mM Tris pH 7.5, 5 mM MgCl2 and 20 μg/ml RNase A). Finally, 223 samples were acquired and analyzed by using BD FACS machine and BD FACS Suite 224 software. The experiments were done in triplicates. For analysis outliers were discarded. 225

Statistical analysis 226
The GraphPad Prism®6 software (GraphPad software Inc.) was used for statistical 227 analysis. All experiments were repeated independently three times. IC50 values were also 228 calculated using non-linear regression analysis with GraphPad Prism®6 software. 229

ANME display cytotoxicity against cancer cell lines in vitro 231
MTT viability assay on HEK293T human embryonic kidney cells, MDAMD-231 breast 232 cancer,MCF7 breast cancer and U87 glioblastoma cells were done separately with a 233 range of concentrations of ANME between 1 to 2000 µg/ml. ANME alleviated cell 234 viability in a dose dependent manner. All the experiments were performed in triplicate. 235 IC50 values were calculated by non-linear regression analysis using Graph Pad Prism 236 software as a mean of three reactions that inhibited 50% of the positive control. The IC50 237 values of ANME in HEK293T human embryonic kidney cells, MDAMD-231 breast 238 cancer, MCF7 breast cancer and U87 glioblastoma were 532.1μg/mL, 100.2μg/mL, 239 205.6μg/mL and 42.23μg/mL, respectively ( showed a remarkable increase in SubG1 (Fig. 2 C-D). This indicated that the breast 252 cancer cell lines were highly sensitive to ANME treatment. These results show that 253 different cell types respond differently to ANME, whereas different cancer cell lines are 254 sensitive to a varying degree, the response ranging from low to high cell death, the 255 normal cell lines did not show any significant effect. 256

ANME inhibits tumor cell growth in vitro by induction of apoptosis 257
The antiproliferative potential of ANME was evaluated in HEK293T human embryonic 258 kidney cells, MDAMD-231 breast cancer, MCF7 breast cancer and U87 glioblastoma. As 259 11 shown in Fig 1A IC50 values of HEK293T human embryonic kidney cells, MDAMD-260 231 breast cancer, MCF7 breast cancer and U87 glioblastoma for ANME treatment 261 were 532.1μg/mL, 100.2μg/mL, 205.6μg/mL and 42.23μg/mL, respectively revealing a 262 relatively higher sensitivity of cancer cell lines. Also there was a general SubG1 arrest in 263 the cells. The Ladder assay revealed a time-dependent increase in DNA fragmentation on 264 treatment with ANME as observed on DNA-gel electrophoresis. DNA extracted from 265 cells treated with ANME showed an increased generation of apoptotic DNA fragments as 266 compared with solvent-treated control cells, it also displays a higher sensitivity of the 267 cancer cell lines relative to normal cell lines. suggested that ANME averted UPR response in both Cancer and 293T cells, as judged 282 by inhibition of p-Ire1α. Figure 4 shows the effect of tunicamycin and increasing 283 concentrations of ANME on the expression of phosphorylated Ire1α. The 284 phosphorylation of Ire1α causes activation of UPR and serves as one of the first markers 285 of UPR activation. As is shown in figure, ANME exposure is associated with decreasing 286 concentration of P-Ire1α in all the four cell types (Fig. 4 A-D) reflecting deactivation of 287 the kinase associated IRE1 signaling of UPR. Xbp1s is another downstream marker of 288 the same pathway. Figure 5 shows the effect of tunicamycin and increasing 289 concentrations of ANME on Xbp1 splicing. ANME inhibits the splicing reaction in all 290 the cell types as indicated by reduced expression of XBp1-s. (Fig.5 A-D), the total 291 phosphorylation level of Ire1α protein decreased, with kinetics matching the inhibition of 292 Xbp1s, consistent with a previous report 32 . 293 Next, we evaluated the effect on the PERK arm of UPR. Figure  the RIDD arm of UPR is also decreased. To check whether ANME directly inhibits Ire1α 307 activity, we tested ANME inhibition on RIDD marker BLOC1S1 (BLOS1) which are 308 considered as standard markers for testing RIDD activity. 33, 34 We found that ANME 309 inhibit Ire1α activity in vitro for RIDD (Fig 8 A-C), indicating the role of ANME on in 310 apoptosis is through the RNAse activity of Ire1α in a dose-dependent manner. 311 13

Ire1/xbp1 and PERK/ATF4 and activation of Caspases 313
Following DNA ladder assay and cell cycle analysis, we probed the effect of ANME on 314 caspase and PARP. Western blots with PARP and caspase antibodies were used to probe 315 the effect of the treatment with different cell lines. The results are shown in Figure 9. 316 HEK293T cell lines did not show any apoptotic signals as revealed by the absence of 317 caspase3 and PARP (Fig.9A), however, other cell lines U87MG, MDAMB231 and 318 MCF7 clearly show the onset of apoptosis in a concentration dependent manner (Figure 9  The expression of Xbp1s and ATF4 was also found to be inhibited in a similar manner at 387 mRNA level. The mRNA expression of BLOC1S1, a marker for RIDD activity was 388 found to be increased on treatment with the extracts, owing to the fact that the extract was 389 downregulating RIDD activities as well. This in in line with the studies carried by These 390 results were consistent with the protein markers of UPR signaling and RIDD activity and 391 reflected a dual modification of the signaling by ANME treatment 45 . 392 The unfolded protein response (UPR) serves as an adaptive mechanism to restore 393 homeostasis. When the stress prolongs and resolution becomes difficult, the UPR 394 An important finding of the study is that in Hek293T cells the cell death via apoptosis 402 was not evident. This may be attributed to the fact that in these cells the UPR has being 403 rescued due to ANME and the in mileu proteostasis was restored as depicted from 404 restoration of UPR markers to the basal level, and that no Caspase 3 or PARP cleavage 405 is seen in these cells. The apoptotic death in other cell lines cancerous in nature can be 406 ascribed to the chronic stress conditions developing as a result of inhibition of the arms 407 after drug treatment that in turn increases ER stress intensity to its threshold level 408 initiating apoptosis through repression of antiapoptotic pre-miRNAs 47,48 or alternatively 409 through upregulation of Casp2 49 . In Hek293T cells the UPR has been rescued due to 410 ANME and the in mileu proteostasis was restored as depicted from restoration of UPR 411 markers to the basal level and also no Caspase 3 or PARP cleavage was seen in these 412 cells. A close association of Ire1α activity and cell fate determination has been proposed 413 that provide evidences of Ire1α being molecular switch that facilitates apoptosis during 414 prolonged ER stress. The caspase activation due to inhibition of PERK arm is in line with 415 earlier studies wherein deletion of PERK in MEF cells subjected to prolonged hypoxia 416 has been reported to produce partial restoration of protein synthesis and enhanced 417 activation of caspases, leading to elevated levels of cell death 50