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. 2016 Mar 17;15(9):1288–1294. doi: 10.1080/15384101.2016.1160980

Novel indolyl-chalcones target stathmin to induce cancer cell death

Barbara Wegiel a,b,#, Yiqiang Wang a,c, Mailin Li a,b, Finith Jernigan a,c, Lijun Sun a,c,#
PMCID: PMC4889226  PMID: 26986925

ABSTRACT

Efficacy of current therapies for advanced and metastatic cancers remains a challenge in clinical practice. We investigated the anti-cancer potency of 3 novel indoly-chalcones (CITs). Our results indicated the lead molecule CIT-026 (Formula = C20H16FNO) induced cell death in prostate and lung cancer cell lines at sub-micromolar concentration. CITs (CIT-026, CIT-214, CIT-223) lead to microtubule destabilization, cell death and low cell proliferation, which in part was dependent on stathmin (STMN1) expression. Knockdown of STMN1 with siRNA against STMN1 in part restored viability of cancer cells in response to CITs. Further, CIT-026 and CIT-223 blocked cancer cell invasion through matrigel-coated chambers. Mechanistically, CITs inhibited phosphorylation of STMN1 leading to STMN1 accumulation and mitotic catastrophe. In summary, we have synthetized novel anti-cancer CIT molecules and defined their mechanism of action in vitro.

KEYWORDS: cell death, inodyl-chalcones, mitosis, stathmin

Introduction

Anti-mitotic agents such as taxanes (i.e. docetaxel) and vinca alkaloids (i.e., vinblastine, vincristine) are among the most effective in treating metastatic disease but their benefits are limited by the rapid development of resistance.1,2 As such, there is still unmet need for selective therapeutic agents that not only target cell division and induce early cell death in the primary tumor but also suppress metastatic spread.1

Chalcone is a key functional group appearing in numerous naturally occurring compounds with anticancer activities and in synthetic anti-tumor agents,3,4 yet novel synthetic molecules with defined specificity are still needed.5,6

Dynamics of microtubules is an important aspect of cell growth. Stathmin (STMN1, OP18, Oncoprotein18) is a microtubule destabilizing protein that plays an important role in the anti-mitotic drug response7 as it regulates mitotic spindle dynamics involved in cell proliferation8 and migration/invasion.9,10 STMN1 acts directly on tubulin heterodimers, promoting destabilization and depolymerization of microtubules. Cancer cells utilize STMN1 to accommodate rapid cell division and proliferation. STMN1 stability and phosphorylation are controlled by common cell cycle regulators such as cyclin-dependent kinase-1 (CDK1), Ca2+/calmodulin-dependent protein kinase II (CaMK II), cdc2 kinase, p53 as well as p38 MAPK (downstream of ASK1).11-13 A vast amount of literature converges on a pro-metastatic role of STMN1 in different types of tumor.14,15 STMN1 was shown to drive metastases of Merkel cell carcinoma in response to sT-antigen.15,16 Similarly, STMN1 silencing significantly reduced proliferation and induced apoptosis in response to ruxolitinib.17 Inhibition of STMN1 with small interfering RNA (siRNA) was shown to sensitize cancer cells to 5-FU treatment.18 However the synergistic effects between 5-FU were question in studies of anti-stathmin ribozyme with 5-FU or adriamycin, in which both were shown to be additive.19 Additionally, knockdown of STMN1 blocked tumor growth in vivo in a model of pancreatic cancer.20 Ribozyme targeting of STMN1 was associated with G2/M arrest and apoptosis of Estrogen Receptor+ (ER+) and ER- breast cancer cells.21 However, overexpression of STMN1 blocked invasion of prostate cancer cells 9 and induced growth arrest at the G2/M phase checkpoint in various breast cancer cell lines.22 STMN1 is also a potential target for modulating tumor angiogenesis as it is a regulator of microtubule dynamics, Rho activity and vascular permeability in endothelial cells.23

Traditional treatment with microtubule-targeting agents (MTAs)24 is only active within a short window of mitotic division. However, modulation of the STMN1 pathway by novel small molecules allows for broader targeting of cancer cells in different cell cycle phases while still inducing cell death as the end point. We hypothesized that targeting of STMN1 via modulation of its phosphorylation with small molecules would block disassembly of existing microtubules independently of the cell cycle progression. In this study, we have synthesized, optimized and tested efficacy of a novel class of anti-cancer agents: CITs.

Results

CIT-026, a novel CIT induces cell death in cancer cells

We have designed and synthesized a new class of chalcone derivatives of indole-tetralone (herein referred as CITs). In total, we synthesized and tested 23 CIT compounds that are structurally distinct from known chalcones with anticancer and anti-inflammatory activities.5,25-28 The discussions of structure-activity relationship (SAR) as well as the identifications of lead compounds, including CIT-026, will be reported separately.

To evaluate the efficacy of CIT-026, we used 4 cancer cell lines: PC3 (prostate cancer), A549 (lung cancer), CLR2119 and PAN02 (pancreatic cancer) and applied CITs in the 0.01-10 μM concentrations range. We showed that CIT-026 strongly decreased survival of cancer cells with IC50 at 0.14-0.3 μM dependently on cell type (Fig. 1A-D), with the highest efficiency in PC3 cells (Fig. 1A). Interestingly, the plateau was achieved at ~0.33 μM and higher doses were not more effective (Fig. 1A-D). CIT-026 did not inhibit DNA synthesis as tested by BrDU proliferation (data not shown), yet it showed significant effect on microtubule stability. We compared the effects of CIT-026 with well-known microtubule-stabilizing agent, docetaxel, in PC3 cells on microtubule stability (Fig. 1E). Interestingly, in contrast to stabilization of microtubules upon treatment with docetaxel, there was abnormal formation of mitotic spindle as well as distribution of α-tubulin and microtubules in PC3 cells in response to CIT-026 (Fig. 1E). This data suggest that CITs may destabilize microtubules and thus alter mitotic spindle formation. Further, this correlated with disintegration of β-actin microfilament cytoskeleton, likely due to induction of cell death (Fig. 1F). Importantly, treatment of PC3 cells with CIT-026 lead to increased number of Annexin V-positive cells, suggesting induction of early apoptosis (Fig. 1G).

Figure 1.

Figure 1.

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Synthetic indolyl-chalcone CIT-026 induces cell death and destabilizes microtubules in cancer cells. (A-D) Crystal violet staining of prostate cancer cells (PC3), lung carcinoma (A549), pancreatic (CLR2119, PAN02) cell lines treated with CIT-026 at various concentrations for 72h. Cells were stained with crystal violet and absorbance corresponding to number of alive cells was evaluated at 562 nm. Data are representative for n = 6. Averages ± SD are shown. p < 0.01 for concentrations ≥ 0.33 μM. IC50 is indicated on the graph for each cell line. (E) Immunostaining analyses of β-actin and α-tubulin in PC3 cells untreated (control) or treated with DMSO, CIT-026 or docetaxel (20 nM) for 24h. Note that the effect of CIT-026 is opposite to taxol. (G) Annexin V staining was applied to test for apoptotic cell death at 24 h. % of gated cells as measured by flow cytometry is shown. Averages ± SD are shown. **p < 0.01.

CIT-026 derivatives are efficient in inducing cancer death

Among the 23 CIT compounds we tested in cancer cell survival assay, we showed that CIT-223 and CIT-214 are similarly efficient as CIT-026 in both PC3 and A549 cell lines (Fig. 2A-B). We identified the most efficient structure of CIT-223 with –CH2CH3 as a side chain as compare to CIT-026 with –CH3 and CIT-214 with –H. Similar inhibition of cell growth and induction of cell death were achieved in A549 and PC3 cells upon treatment with CITs (Fig. 2A-B).

Figure 2.

Figure 2.

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Cell death and STMN1 expression in response to treatment of cancer or normal cells with CIT-026 derivatives. (A-B) Crystal violet staining of PC3 cells (A) or A549 cells (B) treated with 0.01–5 µM CIT-026, CIT-223, CIT-214 for 72 h. n = 4. p < 0.05 for dose ≥ 0.33 μM. Absorbance corresponds to the number of alive cells. Averages ± SD are shown. IC50 PC3, for CIT-026: 0.147 μM, CIT-223: 0.106 μM, CIT-214: 0.449 μM. A549, for CIT-026: 0.208 μM, CIT-223: 0.113 μM, CIT-214: 0.271 μM. (C) STMN1 immunostaining in PC3 cells treated for 24 h with CIT-026 and 2 derivative molecules: CIT-223 and CIT-214. (D) Immunoblotting with antibody against STMN1 in PC3 cells treated with 1 μM CIT-026 (0.01, 0.1, 1 μM) for 30 min or 1h. n = 3. (E) MitoSOX levels corresponding to the mitochondrial derived ROS were measured in the PC3 cells treated with 1 μM CIT-026, CIT-223 or CIT-214. % of gated cells are shown. Data were acquired by flow cytometry after 30 min treatment with compounds.

STMN1 is a target of CIT compounds

Since CIT-026 treatment lead to destabilization of microtubules, we hypothesized that mitotic catastrophe is a part of the mechanism behind the effects of CITs in cancer cell lines. We applied RT2 profiler PCR array to identify specific pathways that were affected by CIT-026 and found 2 genes elevated >4-fold after treatment: adrenomedullin and stathmin (STMN1). Since STMN1 is major mitotic regulator and was affected by CIT in vitro, we focused our study on this protein. We showed accumulation of STMN1 protein expression in cells after treatment with CIT-026, CIT-214 or CIT-223 by immunofluorescence at 24 hours (Fig. 2C). Further, we detected very early (30 min-1h) stabilization of STMN1 in PC3 cells treated with CIT-026 by immunoblotting (Fig. 2D). Importantly, none of the compounds increased ROS levels (Fig. 2E) or influence mitochondria activity as measured by MTT assay (data not shown), suggesting an oxidative stress-independent mechanism of their action.

STMN1 is required in part for the activity of CIT small molecules in vitro

To evaluate the role of STMN1 in mediating anti-tumoral effects of CIT compounds, we employed siRNA against STMN1 in PC3 cells. We confirmed successful knockdown of STMN1 in PC3 cells 48 hours post-transfection with siRNA against STMN1 in both untreated and CIT-026 treated cells (Fig. 3A). Accelerated cell death in response to CIT-026 was attenuated in absence of STMN1 (Fig. 3B). Similar observation was made in PC3 cells transfected with siRNA against STMN1 and treated with CIT-214 or CIT-223 (Fig. 3C-D). Further, we assessed whether decreased levels of STMN1 in PC3 cells transfected with siRNA against STMN1 influenced CIT-026-mediated microtubule destabilization during mitosis. CIT-026 blocked number of cells undergoing mitosis (P-Histone H3) and those presented with normal mitotic spindles (α-tubulin) (Fig. 3E-F). Microtubule destabilization and aberrant mitotic spindle as well as number of P-Histone H3 positive cells in response to CIT-026 were in part reversed in the absence of STMN1 (Fig. 3E-F), suggesting that STMN1 may be one of the critical targets for CIT-026 and responsible in part for the effects of CITs in PC3 cells.

Figure 3.

Figure 3.

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Role of STMN1 in mediating the effects of CIT-026. (A) Immunoblotting with antibody against STMN1 in PC3 cells transiently transfected with siRNA against STMN1 or control siRNA. Efficiency of transfection was tested after 48h and additional 24h after treatment with 1 μM CIT-026. (B-D) Crystal violet staining of PC3 cells transfected with siRNA against STMN1 or control siRNA for 48 h as in (A)and treated with 1 μM CIT-026 (26), 0.1 μM CIT-223 or 1 μM CIT-214 for 72h. **p < 0.01, *p < 0.05. n = 4. (E-F) Immunofluorescence staining with antibody against P-HH3 (green) and α-tubulin (red) in PC3 cells transfected with siRNA against STMN1 or control siRNA for 48h and treated with 1 μM CIT-026 for 48h. Arrows indicate mitotic cells. Number of P-HH3 per FOV is quantified in F. ***p < 0.001. **p < 0.01.

Expression of STMN1 in cancer biopsies

Other studies indicated high expression of STMN1 in cancer samples.29 Analysis of Geo profiles 30 showed high STMN1 mRNA levels in patient samples from advanced and metastatic prostate cancer (Fig. 4A). STMN1 is also strongly associated with microtubule stability and regulates invasion and migration of cancer cells,9 we also investigated whether treatment with CITs affected cancer cell invasion. CIT-026 and CIT-223 strongly inhibited invasion of PC3 cells (Fig. 4B-C) while CIT-214 accelerated migration of PC3 cells through matrigel-coated chambers to the serum chemoattractants as compared to the untreated cells. This data suggest CITs specificity in regulation of migration/invasion versus cell death/growth.

Figure 4.

Figure 4.

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Invasion of cancer cells in response to CIT-026 and its derivatives. (A) Geo profiles data for STMN1 expression in normal prostates (Normal), prostate tissue adjacent to tumor (Adjacent), prostate cancer (PCa) and metastatic cancer samples (Mets). ***p < 0.001, **p < 0.01, *p < 0.05. (B-C) Quantification of number of invading A549 cells after treatment with 0.01–5μM CIT-026, CIT-223, CIT-214 or DMSO for 72 h. n = 4. p < 0.05. Representative pictures of lower part of the invasion chambers were stained with crystal violet for presence of invading cells. ***p < 0.001 treated vs. DMSO.

Phosphorylation of STMN1 is blocked by CIT molecules

Our above preliminary data suggest that CITs lead to early stabilization of STMN1 and thus affect cancer cell viability. We further tested whether CITs influence stabilization of STMN1 by regulating its phosphorylation status. STMN1 can be phosphorylated by proline-directed kinases such as mitogen-activated protein kinase and cyclin-dependent protein kinase and by cAMP-dependent protein kinase.31 We showed that CITs diminished phosphorylation of STMN1 on Ser16, which is associated with higher expression and activity of STMN1 and its effects on microtubules stability (Fig. 5A-C).

Figure 5.

Figure 5.

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STMN1 phosphorylation in the presence of CIT-026. (A-C) Immunoblotting with antibody against P-STMN1 (Ser16) and STMN1 in A549 or PC3 cells untreated or in the presence of DMSO (DM), CIT-026 or its derivatives (CIT-232, CIT-214, CIT-223) for 30 min or as indicated. Data are representative for at least 2–3 independent experiments.

Whether CITs may directly block phosphorylation of STMN1 by effecting its interaction with kinases thus leading to low phosphorylation of STMN1 and destabilization of microtubules and mitotic catastrophe will be addressed in our future studies.

Discussion

In this study, we synthetized and tested novel derivatives of chalcones molecules with anti-cancer activities. We reported high efficacy of these molecules in inducing cell death of prostate and lung cancer cell lines. Despite the difference in structures (CIT-026: R-CH3, CIT-223: R-C2H5, CIT-214: R-H), all CITs had similar pro-apoptotic activity in vitro. Among them however, CIT-214 was the most toxic to normal cells (data not shown) and induced invasion of cancer cell through matrigel-coated chambers. CIT-223 and CIT-026 not only induced cell death but efficiently blocked invasion of cancer cells. This may indicate that the indole NH moiety is not an optimal structure for this class of compounds for wider anti-tumoral activities, including both apoptosis and invasion.

CITs induced mitotic catastrophe in cancer cells via increased abundance of STMN1, which destabilizes microtubules and disrupts cell cycle. STMN1 levels are strongly associated with cancer progression (32,33, Fig. 4A-B) and its expression and stability are regulated by various mechanisms including phosphorylation of STMN1.13 We identified STMN1 to be required for anti-cancer effects of CITs. Low phosphorylation of STMN1 at Ser16 leads to destabilization of microtubules and mitotic catastrophe. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of STMN1 to bind to and sequester tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules.34 Interestingly, phosphorylation profile of STMN1, expression of STMN1 and clinicopathological characteristics correlated with otucomes of breast cancer patients.35 High STMN1 levels or phosphorylation of STMN1 on Ser25 or Ser38 were associated with poorer survival, while high phosphorylation of STMN1 on Ser16 or Ser63 correlated with better prognosis.35

Our data suggest that CITs block STMN1 phosphorylation on Ser 16 to stabilize its function. However, we did not identify which phosphatase activation or kinase blockage is necessary for CITs effect on STMN1 phosphorylation. We showed that STMN1 is necessary in part for anti-cancerous effects of CITs. Knockdown of STMN1 in cancer cell lines reversed in part CITs-induced cell death. Further, cells lacking STMN1 in a presence of CIT-026 proliferate better and showed higher number of P-Histone H3 positive cells as compared to the control scramble siRNA treated with CIT-026. We showed lower number of mitotic figures in untreated cells with knockdown of STMN1 as previously reported. However, overall knockdown of STMN1 decreased efficacy of CIT-026. Recent studies by Wang et al showed that after treatment with paclitaxel or vincristine, STMN1-silencing shRNA-transfected Eca109 and TE-1 cells were more likely to enter G2 but less likely to enter mitosis than control cells.36 Efficacy of tested CITs is independent of the panel of mutated oncogenes or tumor suppressors or cancer cell type, which allows for the broad treatment based on the fact that STMN1 is critical for cell division. Our future studies aim to optimize the CITs to decrease their toxicity in normal cells as well as reach lower IC50. Further, we will establish their efficacy in vivo in mice xenografts.

In summary, we showed high efficacy of a novel class of CITs against prostate and lung cancer cell lines. Finally, we identified STMN1 and microtubules stability as valid mechanisms for anti-tumoral effects of CITs.

Materials and methods

CITs compounds synthesis and treatment

The CIT compounds were synthesized by the aldol condensation between a tetralone and an indole aldehyde.26 Details of reaction conditions, isolations, and characterization data of CIT-026, 214, and 223 are provided in the supplementary material online. All new compounds were characterized by 1H NMR and mass spectra and their purity was >90% by reverse phase HPLC analyses. CITs were diluted in DMSO at 25 mM and then diluted in culture medium at 0.001–10 μM final concentrations. All CITs were soluble and did not precipitate in the medium.

Cell culture

PC3 (kindly provided by Dr. Balk laboratory), PANC02 and CRL2119 (kindly provided by Dr. Seth laboratory), A549 (ATCC) cell lines were cultured according to manufacturer's protocols. Briefly, PC3 were cultured in RPMI medium supplemented with 10% FBS (Atlanta Biologicals), PANC-2, A549 and CRL2119 were cultured in DMEM High medium supplemented with 10% FBS (Atlanta Biologicals).

Crystal violet staining

Cells were seeded at 5 × 103 concentration in 96 well plates and treated with CITs or DMSO (0.001–10 μM) for 72 h. Culture medium was removed and cells were stained with crystal violet solution (Sigma-Aldrich) for 15 min shaking at room temperature. Crystal violet was then removed and cells were repeatedly washed in water. Attached cells stained with crystal violet were dried and then resuspended in 10% acetic acid. Absorbance was measured at 562 nm. The intensity of staining corresponded directly to the number of alive cells.

Annexin V staining

PC3 cells were seeded at 1 × 105 concentration in 6 well plates 24 hours before experiment. Cells were treated with CIT-026 for 24 hours. Cells were harvested and apoptosis rate was measured using Annexin V-FITC staining according to manufacturer's protocol (BD Biosciences, San Jose, CA).

Immunostaining

Immunofluorescence and immunostaining on cell lines and tumor samples was performed as previously described.37 Anti-mouse STMN1, anti-P (Ser10)-Histone H3, and α-tubulin antibody were from Cell Signaling. β-actin antibody was from Sigma-Aldrich. Cells were treated with CIT-026 (1 μM) or DMSO for 48h and after immunofluorescence staining, the number of P-Histone H3 was quantified per filed of view at 40x magnification using Zeiss Axiovert Microscope.

Immunoblotting

Snap-frozen tissue samples were homogenized in ice-cold tissue lysis buffer (250 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM Tris-HCl [pH 7.5]) containing the protease inhibitor cocktail Complete Mini (Roche). Immunoblotting was performed as previously described.39 Antibodies: P-STMN1 (Ser16) (Cell Signaling), STMN1 (Cell Signaling), β-Actin (Sigma) were used at 1:1000 dilution.

Mitosox ROS assay

MitoSox Red mitochondrial superoxide indicator was used to measure ROS generation in PC3 treated with CITs. Briefly, PC3 cells were treated with CITs for 30 min and loaded with 5 μM MitoSox solution and incubated for 10 min in dark at 37°C. Cells were harvested immediately and fluorescence was measured by flow cytometry.

Transfection with siRNA

siRNA against STMN1 and scramble siRNA were from Darmacon (ONTARGET plus siRNA). PC3 cells were transiently transfected with siRNA against STMN1 or scramble siRNA using Lipofectamine 2000 (Invitrogen) as previously described.38 Twenty four hours post-transfection, cells were split to 96 well plates and treated with CITs for 72h. To confirm successful knockdown, we measured STMN1 levels by immunoblotting.

Invasion chambers

The invasion of A549 cells was measured using Transwell chambers (Chemicon, Millipore, CA) according to the manufacturer's protocol. Briefly, A549 cells were treated with CITs at 1 μM in 500 μl of DMEM medium without FBS for 24 hours. Medium in the upper chamber was serum-free while medium in the lower chamber contained 10% FBS as a source of chemoattractants. Cells that passed through the Matrigel-coated membrane were stained and photographed after 24 hours of incubation. Absorbance was measured at 562 nm by ELISA reader after dissolving of stained cells in 10% acetic acid.

RT2 profiler PCR array and Geo Profiles

RT2 profiler PCR array Human Cancer Pathway was purchased from SA Biosciences. RNA from PC3 cells treated with DMSO or CIT-26 was isolated and prepared for RT-PCR following manufacturer’ protocol. Analysis was performed online using the software provided by SA Biosciences. The upregulated genes were further confirmed using real time PCR.

GEO profiles from 18 normal prostatic tissues (without any pathological alterations), 62 tissues adjacent to tumors, 64 primary tumors and 24 metastatic samples were obtained from patients with prostate cancer as described in previously published data.30 Specifically, 24 metastatic biopsies were derived from 4 patients with prostate cancer metastases to the liver, lymph nodes, kidney, lung and adrenal glands.30 STMN1 expression was analyzed.

Statistical analyses

Statistical analyses were performed using GraphPad Prism. Data are presented as the mean ±SD and are representative for at least 3 independent experiments. ANOVA or Student T test was used for estimation of statistical significance for the experiments (p < 0.05).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Supplementary Material

Supplemental Files

Abbreviations

CDK

cyclin-dependent kinase

CIT

indoly-chalcone

ER

Estrogen Receptor

FU

fluorouracil

MD

molecular dynamics

STMN1

stathmin

siRNA

small interfering RNA

Funding

This work was supported by NCI 1R21CA169904-01 and startup funds from Department of Surgery BIDMC (BW) and Seed Fund (LS) funds from Department of Surgery at BIDMC.

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