A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2

Abstract

Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.

Keywords: UBXN2A; chemotherapy; mortalin-2; p53; veratridine.

Figure 1. The heat shock protein mot-2 plays a critical role in tumorigenesis

Equal amounts of tumor tissue lysates from 48 patients with colon cancer (A) and 55 patients with breast cancer (B) along with matched adjacent normal tissue lysates were probed with anti-mot-2 and anti-actin antibodies followed by quantitation and normalization of signals. A–B. These two diagrams show densitometry-quantified signals of mot-2 expression in tumor tissue versus normal adjacent tissue. The expression of mot-2 in the tumor tissues of patients diagnosed with colon cancer (A) and breast cancer (B) was significantly higher as compared to normal tissues (Colon tumor: R² = 0.46 and P < 0.001, Breast tumor: R² = 0.07 and P < 0.05, correlation coefficient test for each pair of variables). C. Overexpression of mot-2 in colon cancer is gender-dependent. Higher numbers of male patients show overexpression of mot-2 as compared to female patients. D. Overexpression of mot-2 in breast cancer is age-dependent. The statistical analysis shows that a relatively higher number of young female patients showed over-expression of mot-2 levels. E–H. Profiling of grade- and stage-dependent of tumors with colon (E and G) and breast (F and H) cancer showed that patients with mot-2 overexpression have been diagnosed with a higher grade and stage of tumors, indicating poorer survival. These clinical results confirm mot-2 as a potential target for the treatment of cancers and a promising prognostic factor.

Figure 2. Induction of UBXN2A slows the growth of a colon cancer tumor ex vivo and in a mouse xenograft model by 50%

A. A Tet-on regulated inducible HCT-116 colon cancer cell line was established successfully. 48 hours incubation with Doxycycline (DOX, 10 mg/ml) induces an equal expression of GFP-empty (EMP) or GFP-UBXN2A in HCT-116 cells. Images were taken using an inverted fluorescent microscope. B. WB showing the expression of GFP-empty and GFP-UBXN2A proteins in HCT-116 cells when treated with DOX for 48 hours. An anti-GFP antibody was used to detect the levels of GFP alone or of GFP-UBXN2A fusion proteins, while GAPDH was used as a loading control. Optical density of bands were measured by Image Studio software C. Inducible cells (GFP-EMP or GFP-UBXN2A) were treated with DOX for the indicated times. Cells were then stained with PerCP-Cy5.5 Annexin V antibody, and a total of 10, 000 gated events were analyzed by flow cytometry. DOX-induced UBXN2A expression in 48 and 72 hours showed a significant increase in annexin binding in those cells. The data is shown as mean ± SEM of three independent experiments in trplicate (n = 3) where *p < 0.001 using Tukey's modified student's t-test. D. Inducible cells (GFP-EMP or GFP-UBXN2A) were treated with DOX for 48 hours, followed by staining with Alexa Fluor 546 anti-caspase-3 antibody. Cells were analyzed using flow cytometry and collected data was normalized with GFP-EMP (+DOX). The DOX-induced UBXN2A expression significantly increased caspase-3 levels (n = 3, *p < 0.05). E. Inducible cells (GFP-EMP or GFP-UBXN2A) were treated with DOX for the indicated times. Immunofluoresence of p21 was performed. Intensity of fluorochrome signals from a confocal microscope were analyzed by the Image J (NIH, Bethesda, MD) program and plotted. The data was normalized with GFP-EMP (+DOX). The expression of UBXN2A significantly increased levels of p21 (n = 100, *p < 0.001). F–G. HCT-116 cells expressing GFP-EMP and GFP-UBXN2A under DOX were treated with 5-FU (100 μM) for 48 hours. Cells were then stained with Alexa Fluor 546 anti-caspase-3 (F) or anti-cleaved PARP (G) antibodies, followed by flow cytometry analysis. The induced UBXN2A significantly enhanced 5-FU-induced increase in caspase-3 and cleaved PARP levels (n = 3, *p < 0.05). H. Inducible HCT-116 cells were incubated with DOX and 5-FU for 48 hours, stained with Sytox Red, and then the population of dead cells was analyzed using flow cytometry. UBXN2A significantly enhanced 5-FU-induced cell death (n = 3, *p < 0.05). I–J. 1 × 107 Tet-on inducible cells were injected subcutaneously into the lower flanks of nude mice. The animals with palpable tumors (< 5 mm3) for early-stage tumor response were divided into two groups after injection to be fed with a standard diet (controls) or a DOX-containing diet (625 mg/kg). Tumor volumes in mm3 were determined with digital calipers by the formula Volume = (width) 2 × length/2 every second day for 40 days. K. Expression of GFP-empty or GFP-UBXN2A proteins in dissected xenografts confirmed successful tumor responses to DOX after 40 days. L. Growth of tumors with and without DOX on day 40. Treatment with DOX significantly decreases tumor size and weight by more than 50%. The data is shown as mean ± SEM of 9 mice (n = 4 for control and n = 5 for DOX) where *p < 0.05 using Tukey's modified student's t-test.

Figure 3. Veratrine and its purified form VTD increase UBXN2A level in vitro and in vivo

A. A cell-based screen was conducted in search of compounds that induce the expression of the UBXN2A gene. The 3.9 K base of DNA upstream from the UBXN2A gene on human chromosome 2, including endogenous promoters and necessary enhancers as well as untranslated exon 1, was cloned into MCS-mGL.1, a Gaussia luciferase vector, and transiently transfected into HCT-116 colon cancer cells. We used empty MCS-mGL.1 for background expression. This cell line was used to screen over 1800 FDA (Food and Drug Administration) approved drugs, synthetic compounds, and natural products. A glow luciferase activity assay was conducted in triplicate. We found 12 potential candidates in the initial screen, which were confirmed again by the luciferase assay. B–C. 40 μM Veratrine sulfate (an unpurified form of VTD) resulted in a ∼twofold increase in luciferase activity when compared to control. D. WB experiments showed incubation of HCT-116 with Veratrine for 24 hours leads to up-regulation of UBXN2A, while Veratrine has no effect on p47 (UBXN2C), another member of UBXD family. GAPDH was used as a loading control. Staurosporine, as an alkaloid, was used as a negative control. E. IP injection of Veratrine (0.125 mg/kg) to C57Bl/6N mice for 28 days showed a selective upregulation of UBXN2A in small intestine and colon tissues, but no changes were observed in the liver of the same animals. F. VTD, a naturally occurring plant alkaloid of steroidal structure, is a component of Veratrine mixture. G. HCT-116 cells were treated with VTD (20 and 40 μM) and cell lysates were subjected to WB. GAPDH was used as a loading control. VTD increases UBXN2A protein levels in a dose-dependent manner.

Figure 4. Induction of apoptosis and in vitro cytotoxicity by VTD in human cancer cell lines with different statuses of p53 and mot-2

A–D. Cells were treated with VTD for 24, 48, and 72 hours. After incubation, dead cells were washed off, and the remaining attached cells were calculated by counting in five random fields using the Axiovert 200 M inverted microscope and AxioVision software (cell detachment assay). The % of cells attached was calculated as % of control cells. VTD significantly decreases the % of cells attached in a dose-dependent manner in cancer cells with WT-p53 (A: HCT-116, B: LoVo and C: U2OS) but not with mutant p53 (D: SW480). The data in Panel D suggest Veratridine might promote cell proliferation. However, a strict statistical analysis rejects any significant changes in SW-480 cells. The data is shown as mean ± SEM of three independent experiments (n = 3, *p < 0.05). E–K. Cells were plated for 5 days, and the colonies of cells were treated with different concentration of VTD. The colonies of viable cells were stained with crystal violet dye and absorbance, as an index of measurement of colony forming units, was read at 562 nm (clonogenic survival assay). VTD induced a significant decrease in cell viability in a p53- and differentiation grade manner in HCT-116 poorly differentiated cells (E), HCT-116 p53^+/− (F), HCT-116 p53^−/− (G), LoVo well-differentiated cells (H), SW-480 (K), and two non-colon cancer cells: HepG2 (I) and U2OS (J) The data is shown as mean ± SEM of three independent experiments (n = 3, *p < 0.05). L. Determination of endogenous levels of mot-2, UBXN2A, HSC70, and p53 proteins in various cancer cell lines using WB. M. HCT-116 cells were treated with VTD (10 and 30 μM) and cytoplasmic and nuclear fractions were subjected to WB. HSC70 and Orc-2 antibodies were used as cytoplasmic and nuclear markers, respectively. VTD increases p53 in both cytoplasm and nucleus compartments in a dose-dependent manner.

Figure 5. VTD functions via the UBXN2A-mot-2-p53 axis

A. WB confirmed the presence and the absence of p53 protein in HCT-116 p53^(+/+) and HCT-116 p53^(−/−) cancer cell lines, respectively. B–D. The effect of VTD on the viability of poorly-differentiated (HCT-116 p53^(+/+) and HCT-116 p53 ^(−/−)) and well-differentiated (LoVo) colon cancer cell lines was determined using an MTT assay. VTD's cytotoxic effect on all three cell lines was dose dependent in (B) 24 hours, (C) 48 hours, and (D) 72 hours. As compared to the HCT-116 p53^(+/+) cells, HCT-116 p53^(−/−) cells showed more resistance to VTD's effects, whereas well-differentiated (LoVo) colon cancer cells showed the highest sensitivity to VTD. E. LoVo cells were stably silenced for UBXN2A using a UBXN2A-shRNA along with a scrambled shRNA. Viability of UBXN2A-silenced cells was found to be significantly higher as compared to control cells upon treatment with VTD. F. In another set of experiments, UBXN2A-silenced cells were treated with VTD (100, 200, and 300 μM) for 24 hours. Cells were then labelled with Sytox Red followed by flow cytometry analysis. Results showed silencing of UBXN2A significantly decreases cell death in response to VTD. G. SW48 well-differentiated colon cancer cells with WT-p53 were treated with MKT-077 (5 μg/ml), a mot-2 inhibitor, along with VTD for 72 hours. A clonogenic survival assay showed VTD signifcantly decreases the colony number of SW-48. However, preincubation with MKT-077 neutralizes the cytotoxic effect of VTD. The data is shown as mean ± SEM of three independent experiments (n = 3) in triplicate where *p < 0.05 using Bonferroni's modified student's t-test. H. This flowchart recapped the sequence of proteins which activate and function upon VTD exposure to decrease cell viability.

Figure 6. Synergistic inhibitory effects of VTD and etoposide or 5-FU chemotherapeutic drugs on the growth of human colon cancer cells

A–C. HCT-116 and D–F. LoVo cells were treated with VTD (10- 300 μM) for 24, 48, and 72 hours. A suboptimal dose of Etoposide (ETO) (5 μM) was added to cells for the last 24 hours only. The viability of cells was determined as % of control (untreated cells) using an MTT assay. The chemotherapeutic drug ETO significantly enhanced the VTD-induced decrease in cell viability of both poorly differentiated (HCT-116) and well-differentiated (LoVo) colon cancer cell lines in a time-dependent manner. G–H. HCT-116 (G) and LoVo (H) cells were treated with VTD (10–200 μM) and a suboptimal dose of ETO (5 μM) for 24 hours. Cells were then stained with Annexin V apoptotic marker. ETO significantly sensitizes both poorly and well-differentiated colon cancer cells to VTD even at 30 μM. I–L. HCT-116 and LoVo cells were treated with different doses of 5-FU (5–100 μM, I-J) and ETO (1–50 μM, K-L) along with VTD (30 μM and 100 μM respectively) for 24 hours. MTT assays showed the treatment with 5-FU and ETO with VTD decreased cell viability at much lower drug doses particularly in LoVo cells. Results were analyzed using CalcuSyn software to calculate the combination index (CI) to confirm and quantify the synergism observed with combination therapies (S6 and S7 and Supplementary Tables 4 and 5). The data is shown as mean ± SEM of three different experiments (n = 3) where *p < 0.001 using Bonferroni's modified student's t-test.

Figure 7. Sensitization of colon cancer cells to a long-term suboptimal dose of 5-fluorouracil exposure when combined with VTD

A–D. (A) HCT-116, (B) LoVo, (C) SW-480, and (D) U2OS were treated with VTD (10–300 μM) for 10 days. Colony formation assays showed VTD effectively reduces cell viability in a cell type-dependent manner. E–G. (E) HCT-116, (F) LoVo, and (G) U-2OS were treated with suboptimal doses of 5-FU and VTD (50 and 100 μM) for 10 days. Clonogenic survival assays revealed an intermediate-dose of VTD significantly potentiates the standard chemotherapy used at very low dose (1–5 μM 5-FU). H–I. A series of flow cytometry assays using an antibody against the CD44 cancer stem cell marker illustrated that VTD can target CD44+ cancer stem cells and eventually eliminate them in a dose-dependent manner, implicating VTD as a potential cancer stem cell-targeting therapy. J. Tumor tissue lysates from 48 patients with colon cancer alongside the matched adjacent normal colon tissue lysates were probed with anti-UBXN2A and anti-Actin antibodies, followed by quantitation and normalization of signals. UBXN2A expression shows a marked downregulation in ≥ 50% of patients with colon cancer. UBXN2A expression levels may have a correlation with the stages of colon cancer as several patients at stage III had low level of UBXN2A (inset). (I) The proposed mechanism action of VTD. VTD increases the expression of UBXN2A, which releases the p53 from mot-2′s sequestration and, together with standard chemotherapy, can cause an effective tumor suppression.