Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of e2f

ABSTRACT Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment.

Triptolide, a natural compound isolated from the Chinese herb _Tripterygium wilfordii,_ is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named

Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer

and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of

colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate

dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb)

binding, and proteins levels of regulator of G1–S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer

cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than

doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at

higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also

show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon

cancer in multiple pre-clinical models. SIMILAR CONTENT BEING VIEWED BY OTHERS A NOVEL RUTHENIUM COMPLEX WITH 5-FLUOROURACIL SUPPRESSES COLORECTAL CANCER STEM CELLS BY INHIBITING AKT/MTOR

SIGNALING Article Open access 16 December 2023 UTIDELONE INHIBITS GROWTH OF COLORECTAL CANCER CELLS THROUGH ROS/JNK SIGNALING PATHWAY Article Open access 01 April 2021 ALBENDAZOLE INHIBITS

COLON CANCER PROGRESSION AND THERAPY RESISTANCE BY TARGETING UBIQUITIN LIGASE RNF20 Article 26 January 2024 MAIN Colorectal cancer is the third most common cause of cancer-related deaths in

the United States.1 Despite significant progress in the prevention, early diagnosis and management of colorectal cancer, over 50 thousand patients died from this disease in 2012.1

Oxaliplatin is the latest addition to the armamentarium against colon cancer and its addition to the original adjuvant therapy regimen of 5-FU and leucovorin improves the 5-year disease-free

survival of stage II and III patients from 67% to 73% with minimal improvement in overall survival.2 Those presenting with metastatic disease have a grim prognosis with median survival

ranging from 15 to 20 months.3 Thus, there is an urgent need to develop effective therapies for this formidable disease. Triptolide, a natural compound isolated from the Chinese plant

_Tripterygium wilfordii_, has anti-inflammatory and anticancer properties. Previously, we and others have shown that triptolide is effective against multiple types of cancer. Triptolide

induces cell death in pancreatic cancer cells and markedly reduces the growth and locoregional spread of pancreatic tumors in animal models.4 We have also shown that triptolide is effective

against osteosarcoma,5 lung cancer,6 cholangiocarcinoma,7 and neuroblastoma.8 Others have shown that triptolide is effective against melanoma,9 gastric,9 breast,9 and colon cancer.10, 11 We

have now synthesized the water soluble analog of triptolide, named Minnelide, and have evaluated it extensively in multiple animal models and scenarios of pancreatic cancer12 with

encouraging results. Whether Minnelide is efficacious against colon cancer is not known. Although our previous studies suggest that the downregulation of HSP70,4 Mcl-1,13 and Sp114

contribute to triptolide-induced cancer cell death, the exact mechanism of action of triptolide remains elusive. Our previous work also suggests that triptolide can induce multiple types of

programmed cell death, inducing apoptosis and autophagy depending on the cancer cell type.15 In the current study we demonstrate that Minnelide is effective against colon cancer in the

xenograft and liver metastasis model. Mechanistically, we show that at low concentrations triptolide induces cell death by apoptosis, but at higher concentrations it induces cell cycle

arrest. Furthermore, our studies reveal that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F. Our data also suggest that triptolide

downregulates E2F activity by potentially modulating events downstream of its DNA binding. MATERIALS AND METHODS REAGENTS Colorectal cancer cell lines HCT116 and HT29 were purchased from

ATCC (Manassas, VA, USA). Human colon epithelial cells (HCEC) were a generous gift from Professor Shay.16 Triptolide, Guava Nexin Apoptosis kit, Guava cell cycle reagent, phosphatase

inhibitor cocktail II were purchased from EMD Millipore Chemicals (Billerica, MA, USA). DMSO was purchased from Sigma Aldrich (St Louis, MO, USA). Cell Counting Kit-8 was from Dojindo

Molecular Technologies (Rockville, MD, USA). FuGENE-HD transfection reagent, Dual-Luciferase Assay system, Caspase-Glo 3/7 assay and CytoTox 96 Non-Radioactive Cytotoxicity Assay were from

Promega (Madison, WI, USA). QuantiTect SYBR Green PCR kit was from Qiagen (Valencia, CA, USA). Cignal E2F Reporter kit was from SABiosciences, Qiagen. TRIzol Reagent was from Life

Technologies (Great Island, NY, USA). Phosphate-buffered radio immunoprecipitation assay (RIPA) buffer, beta-glycerophosphate and sodium-pyrophosphate were from Boston BioProducts (Ashland,

MA, USA). Protease inhibitors were from Roche (Mannheim, Germany). Tris-HCL polyacrylamide gels and nitrocellulose membranes were from BioRad laboratories (Richmond, CA, USA). BCA protein

assay kit was from Pierce (Rockford, IL, USA). High Capacity cDNA Reverse Transcription Kit was from Applied Biosystems (Foster City, CA, USA). McCoy’s 5A medium was from Thermo Scientific

(South Logan, UT, USA). For western blotting antibodies against cyclin-dependent kinase 4 (CDK-4), cyclin-dependent kinase 6 (CDK-6), p15, and cyclins A, D1, and E2 were purchased from Cell

Signaling (Danvers, MA, USA). All secondary antibodies as well as streptavidin HRP conjugate used in the study were also purchased from Cell Signaling. Biotin-conjugated Rb antibody was from

Thermo Scientific. Antibody against p21 and biotin-conjugated antibody against E2F1 were from Abcam (Cambridge, MA, USA). Beta-Actin primary antibody was purchased from Santa Cruz

Biotechnologies (Santa Cruz, CA, USA). CELL CULTURE AND DRUG TREATMENT Colorectal cancer cell lines HCT116 and HT29 were cultured in McCoy’s 5A medium supplemented with 10% FBS and 1%

penicillin–streptomycin at 37 °C in a humidified 5% CO2 atmosphere as recommended by ATCC. The immortalized epithelial cell HCEC were maintained with DMEM media (HyClone, Logan, UT, USA)

supplemented with EGF (25 ng/ml, Cell Signaling), hydrocortisone (1 _μ_g/ml, Sigma), insulin (10 _μ_g/ml), transferrin (2 _μ_g/ml), sodium selenite (5 nM) (Gibco), 2% cosmic calf serum

(HyClone), gentamicin (Gibco) and amphotericin B (Corning) in Primaria flasks (BD Biosciences) under 2% oxygen, 5% carbon dioxide and 93% nitrogen. Triptolide was dissolved in DMSO to

provide a stock solution of 1 mg/ml. Cells were treated with triptolide at final concentrations of 0–200 nM in McCoy medium with 5% FBS and 1% penicillin/streptomycin antibiotics.

DETERMINATION OF CELL VIABILITY Cell viability was determined using the Dojindo Cell Counting Kit-8. After treatment with triptolide for the appropriate duration (24–72 h), cell viability

was measured by incubation with 10 _μ_l of the tetrazolium substrate for 1 h at 37 °C; absorbance at 450 nm was measured. ANIMAL EXPERIMENTS All animal experiments were reviewed and ratified

by the Institutional Animal Care and Use Committee at the University of Minnesota. Three different animal models were carried out. For the xenograft model 1 × 106 HCT116 cells were

suspended in matrigel and injected subcutaneously in the right flank of 2-month-old female nude mice. Seven days post implantation, the mice were randomized into following two groups: (1)

control (injected with saline only) and (2) Minnelide (0.42 mg/kg/day). Mice were treated daily and tumor size was measured weekly and tumor volume was calculated as 0.52 × length × width2.

This model was terminated after 30 days. For the liver metastasis model, animals were anesthetized with a mixture of ketamine/xylazine (100 mg/kg and 10 mg/kg). 1 × 106 HCT116 cancer cells

suspended in matrigel were injected under the splenic capsule at laparotomy and wound was closed using absorbable sutures and surgical staples. Five days post injection mice were randomized

into control (injected with saline only) and Minnelide (0.42 mg/kg/day) groups. Four weeks after the start of the treatment animals were euthanized and liver metastases were dissected out.

The number and cumulative weight of visible liver metastases was compared between two groups. In a separate liver metastasis model, animal survival was used as the end point. In this model 2

× 106 cells were injected under spleen capsule and liver tumors were allowed to develop. Four weeks post implantation, animals were randomized and treated as described above. In accordance

with animal care guidelines, animals were euthanized if they lost >20% of baseline weight, developed excessive ascites, or showed any other signs of failure to thrive. This was considered

death of the animal. The experimentation was terminated 80 days after start of treatment. CASPASE-3 ACTIVITY ASSAY Caspase-3 activity was measured by the Caspase-Glo 3/7 assay from Promega

according to the manufacturer’s protocol. MEASUREMENT OF ANNEXIN-V-POSITIVE CELLS For evaluation of phosphatidylserine externalization (marker of early apoptosis), after appropriate duration

of treatment with triptolide, colon cancer cells were stained with Guava Nexin reagent according to the manufacturer’s protocol and then analyzed (5000 events) on a Guava PCA flow

cytometer. LDH RELEASE ASSAY Necrosis was measured by lactate dehydrogenase (LDH) release assay. In brief, after triptolide treatment, LDH activity in the supernatant and in the whole cell

pellet (after lysing the cells) was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega according to the manufacturer’s protocol. LDH activity in the supernatant when

compared with total LDH content provided the percent LDH release and thus the extent of necrosis. CELL CYCLE ANALYSIS For cell cycle analysis colon cancer cells were serum starved for 12 h

for cell cycle synchronization and then treated with triptolide (0–200 nM) for 12–24 h. After appropriate treatment cells were harvested by trypsinization, washed with PBS, and resuspended

in PBS at a concentration of 106cells/ml. A 100 _μ_l aliquot of this suspension was taken and mixed with 100 _μ_l Guava cell cycle reagent, incubated in the dark for 30 min and then acquired

on BD flow cytometer. LUCIFERASE REPORTER ASSAY FOR E2F E2F activity assay was performed using the Cignal reporter assay kit (Cat no. CCS-003L, SABiosciences). In brief, cells were

transfected with reporter plasmid (200 ng/well) using FuGENE transfection reagent. Appropriate negative control was used in all experiments. Cells were allowed to grow for 24 h before

triptolide treatment for 12 h. Cells were collected after treatment and analyzed using the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s protocol.

QUANTITATIVE REAL-TIME PCR Quantitative real-time PCR for Orc1, Cyclin A-1, and CDC-2 was carried out using primers obtained from Qiagen (QuantiTect primer assay). RNA was isolated from the

different cell lines and from the tumor samples according to the manufacturer’s instructions using TRIzol (Invitrogen). Total RNA (1 μg) was transcribed into cDNA and real-time PCR was

performed using the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions on an Applied Biosystems 7300 real-time PCR system. All data were normalized to the

housekeeping 18-S gene (18S QuantiTect primer assay, Qiagen). WESTERN BLOTTING Protein concentration in cell lysates was estimated using the BCA protein estimation assay (Thermo Scientific).

Lysates were western blotted for Cyclin D1, Cyclin E2, CDK-4, CDK-6, p15, p21, Rb, E2F1, and actin using the western blotting protocol described before.17 IMMUNOPRECIPITATION For

immunoprecipitation 250–500 _μ_g of appropriately treated cell lysates were adjusted to a final volume of 500 _μ_l and pre-cleared with 1 _μ_g of normal rabbit IgG (Santa Cruz

Biotechnologies cat# 2027), and 20 _μ_l of nProtein A Sepharose 4 fast flow (GE Healthcare, cat# 17-5280-01) at 4º C for 30 min on rotating platform. Following incubation lysates were

centrifuged at 1000 g for 30 seconds at 4 ºC, and supernatants were collected and incubated with 1 _μ_g of anti-E2F1 antibody (Invitrogen cat#321400), or 1 _μ_g of anti-Rb antibody (BD

Biosciences cat#554162) on a rotating platform at 4 °C overnight. After overnight incubation, 20 _μ_l of nProtein A Sepharose 4 fast flow was added to lysates and incubated for 2 h at 4 °C

with constant rotation. Lysates were then centrifuged at 1000 G for 30 s at 4 °C and supernatant was discarded. Sepharose pellets were gently washed four times with 1.0 ml RIPA, and after

the final wash pellets were resuspended in 40 _μ_l of (2 × ) Laemmli Sample Buffer (BIORAD, cat#: 161-0737) and boiled for 3 min. Immunoprecipitates were then western blotted and probed for

Rb or E2F1. CHROMATIN IMMUNOPRECIPITATION In all, 2 × 106 HCT116 and HT29 cells were plated in 10-cm dishes. Twenty four hours after plating, the cells were treated with triptolide 50, 100,

and 200 nM for 12hours. Following treatment chromatin was cross-linked, isolated, immunoprecipitated, cleaned, and PCR performed using Pierce Agarose CHIP kit (Cat # 26156) and using

manufacturer’s protocol. E2F1 CHIP grade antibody and target CDC-2 primer were obtained from Millipore (Cat #17-10061). STATISTICAL ANALYSIS Values are expressed as mean±s.e. All experiments

with cells were repeated at least three times. The significance of the difference between the control and each experimental test condition was analyzed by unpaired Student’s _t_-test.

P<0.05 was considered statistically significant. Survival was analyzed by Kaplan–Meier curves and log-rank test. RESULTS TRIPTOLIDE AND MINNELIDE ARE EFFECTIVE AGAINST COLON CANCER _IN

VITRO_ AND IN MULTIPLE MOUSE MODELS The effect of different doses of triptolide on the viability of colon cancer cells was evaluated. As seen in Figures 1a and b, triptolide decreases the

viability of HCT116 and HT29 colon cancer cells in a dose- and time-dependent fashion. To evaluate the effect of triptolide on colon cancer _in vivo_, three different models of colon cancer

were employed. The first was a xenograft model where 1 × 106 HCT116 colon cancer cells were implanted subcutaneously, allowed to grow for a week, and randomized into the control (no

treatment) and treatment (Minnelide 0.42 mg/kg/day) arms. As mentioned previously Minnelide is a water soluble analog of triptolide, which we have shown to be very effective against

pancreatic cancer in multiple animal models.12 Mice were treated for 30 days and the tumor volume was compared between the two groups on a weekly basis. As per University of Minnesota animal

care guidelines the experiment was terminated when the tumor volume exceeded 1 cm3. As seen in Figure 1c, Minnelide treatment reduced the growth of colon cancer xenografts significantly

when compared with the vehicle alone group. As liver metastases of colorectal cancer is a significant clinical problem, we used the liver metastasis model of colon cancer. In this model

HCT116 colon cancer cells were injected under the splenic capsule and allowed to metastasize to the liver. Five days post implantation, the animals were randomized into control (no

treatment) and Minnelide (0.42 mg/kg/day) treatment groups. Mice were treated with the designated agent for 4 weeks, killed, and liver metastases dissected out. The number and weight of

liver metastases was compared between the two groups. As seen in Figures 1d and e, Minnelide treatment reduced the number and the weight of liver metastases significantly when compared with

the control group. Photographs of representative tumor implant in the liver are shown in Figure 1f. The left panel shows the tumor-liver interface at × 10 and the right panel shows the tumor

at higher magnification of × 200. In another independent experiment, we evaluated the impact of Minnelide treatment on survival in the liver metastasis model of colon cancer. In this model

animals were randomized 4 weeks post implantation into control (no treatment) and Minnelide (0.42 mg/kg/day) treatment groups. Animals were then treated daily and followed for any signs of

failure to thrive and >20% weight loss. If these signs were observed the animals were euthanized and this was considered cancer-related death. At the end of the model the median survival

in the two groups was compared. Figure 1g demonstrates the Kaplan–Meier curve demonstrating the survival in the two treatment groups. As seen in Figure 1g, Minnelide treatment significantly

(_P_<0.0001) improved the survival of animals with liver metastases. To evaluate if the effects of triptolide are specific to transformed cells, we evaluate the effect of triptolide on

the viability of normal colonic epithelial cells. As seen in Figure 1h, triptolide does not affect the viability of normal human colonic epithelial cells, even at high concentrations.

TRIPTOLIDE INDUCES APOPTOSIS AT LOW DOSE BUT CAUSES G1 PHASE ARREST AT HIGHER DOSE Once we had established the efficacy of Minnelide in decreasing tumor growth and increasing survival, we

proceeded to examine the mechanism by which triptolide decreases growth of colon cancer cells. We have previously shown that triptolide can induce cell death in pancreatic cancer cells by

multiple mechanisms, including apoptosis and autophagy.15 To evaluate the effect of triptolide on apoptotic cell death in colon cancer cells, the effect of triptolide on Annexin-V (a marker

of early apoptosis) and caspase-3 (effector caspase of caspase-dependent apoptosis) was measured. As seen in Figures 2a–d, triptolide treatment results in an increase in annexin V positivity

and caspase-3 activation in both HCT116 and HT29 colon cancer cells. Interestingly, contrary to previously published data using other cancer cell lines, we did not observe a dose-dependent

increase in apoptosis. The caspase-3 activation and annexin positivity increased up to a triptolide dose of 25–50 nM and then decreased at concentrations above 50 nM. As we have previously

observed that triptolide treatment can lead to programmed cell death by multiple different mechanisms, we evaluated whether triptolide at higher concentrations can induce other forms of cell

death in addition to apoptosis. As seen in Figures 2e and f, triptolide treatment does not induce significant necrosis as measured by LDH release assay. We also evaluated the effect of

triptolide on autophagy by measuring LC3 II levels by western blot and did not observe any change (data not shown). In search for the mechanism by which triptolide induces increased cell

death despite reduced apoptosis at higher dosage, we next looked at the effect of triptolide on cell cycle progression in colon cancer cells. As seen in Figures 3a and b, triptolide

treatment significantly increases the percentage of cells in G1 phase in both colon cancer cell lines tested as compared with control (from ~50% in untreated cells to 80% at 200 nM). This

suggests that at higher concentrations, triptolide induces cell cycle arrest in colon cancer cells. Data from a representative experiment are shown in Figure 3c. TRIPTOLIDE INHIBITS

TRANSCRIPTIONAL ACTIVITY OF E2F, THE KEY REGULATOR OF G1–S TRANSITION We next focused on the mechanism by which triptolide induces G1 cell cycle arrest in colon cancer cells. Activity of E2F

transcription factors (E2F1–3) is crucial to progression of cell cycle from G1 to S phase. Given that triptolide treatment induces a G1 cell cycle arrest, we evaluated the effect of

triptolide on E2F activity using a luciferase promoter assay. As seen in Figure 4a, triptolide treatment results in a dose-dependent decrease in the transcriptional activity of E2F in HCT116

cell line. As E2F1 is the most well understood and characterized member of E2F family of proteins, we focused primarily on this subtype. To further confirm that triptolide inhibits

transcriptional activity of E2F1, we evaluated the effect of triptolide on downstream targets of E2F1, namely cyclin A, Orc1, and CDC-2. As seen in Figures 4b–d, triptolide treatment leads

to a dose-dependent decrease in the expression of cyclin A, Orc1, and CDC-2 in both HCT116 and HT29 cell lines, confirming that E2F1 activity is downregulated by triptolide. Next, we sought

to define the mechanism by which triptolide downregulates the transcriptional activity of E2F1. Phosphorylation of Rb in E2F1-binding pocket by CDK-4 and CDK-6 in conjunction with cyclin D

and CDK-2 in conjugation with cyclin E leads to the release of E2F1, which in turn, can regulate E2F1-dependent genes, and thus regulate the G1 to S phase transition. Activity of CDKs is

further downregulated by p15, p16, and p21. We therefore examined the effect of triptolide treatment on the levels of these proteins. We hypothesized that if triptolide-mediated G1 arrest

was through its effect on the levels of these proteins, triptolide treatment would result in decreased levels of cyclin D, cyclin E, CDK-4, and CDK-6, and increase in levels of p15 and p21.

Both HCT 116 and HT29 lack p16. As seen in Figures 5a and b triptolide treatment does not alter levels of these proteins in a way that will explain G1 arrest at doses 100–200 nM. To further

confirm that triptolide is not inducing G1 arrest by affecting sequestration of E2F1 by Rb, we evaluated the effect of triptolide on Rb-E2F1 binding by co-immunoprecipitation experiments. As

seen in Figures 5c and d, triptolide treatment does not lead to increased binding of E2F1 to Rb. We next evaluated the effect of triptolide on E2F DNA binding by chromatin

immunoprecipitation assay. As seen in Figures 5e and f, at 12 h triptolide treatment does not decrease but conversely increases the E2F1 biding to CDC-2 promoter. Our data taken together

show that triptolide does not decrease E2F1 DNA binding. DISCUSSION In the current manuscript we demonstrate that triptolide, a diterpene triepoxide from a Chinese herb _Trypterigium

wilfordii_, kills colon cancer cells _in vitro_. We also demonstrate that Minnelide, a novel water soluble analog of triptolide, is very effective in inhibiting the growth of colon cancer in

multiple animal models. Mechanistically, our data suggest that although triptolide activates apoptosis in colon cancer cells, at higher concentrations cell cycle arrest is a more prominent

mechanism. Furthermore, we demonstrate that triptolide induces G1–S arrest in colon cancer cells by decreasing E2F1 transcriptional activity at steps downstream of its binding to promoter of

E2F1-regulated genes. To our knowledge, this is the first report demonstrating that triptolide can induce G1–S arrest by modulating E2F1 activity. Finally, the evaluation of a novel water

soluble analog of triptolide, in a clinically relevant liver metastases model of colon cancer, is an additional strength of the current study. Triptolide has been evaluated and shown to be

effective against multiple cancer types including pancreatic cancer,4 neuroblastoma,8 osteosarcoma,5 cholangiocarcinoma,7 breast cancer,9 glioblastoma,18 multiple myeloma,19 and prostate

cancer.20 Triptolide’s efficacy has been tested against colon cancer as well.4, 11 However, detailed evaluation of the mechanism as well as evaluation of a novel water soluble analog in

multiple animal models of colon cancer distinguishes the current study from previous work. One of the major obstacles in the application of triptolide in clinical practice has been its low

solubility in water. We have recently circumvented this hurdle by synthesis of its water soluble analog Minnelide,12 which we have extensively evaluated in pre-clinical models of pancreatic

cancer. Phase I clinical trial evaluating Minnelide in patients with pancreatic cancer are currently under way. In the current study we have shown that Minnelide is very effective in both

subcutaneous xenograft model as well as liver metastasis model of colon cancer. The utilization of a liver metastasis model of colon cancer is pertinent to the evaluation of the efficacy of

any compound against colon cancer, as upwards of 50% patients with colon cancer develop liver metastases during the course of their disease.21 In fact, up to a quarter of patients with colon

cancer present with liver metastases at the time of diagnosis. Although liver resection in these patients with colorectal cancer with liver metastases can offer long-term disease-free

survival, only a fraction of patients have liver disease amenable to resection. Patients who undergo liver resection are still at risk of recurrent disease. Those who have liver metastases

not amenable to resection only have systemic chemotherapy as a treatment option. The fact that Minnelide treatment was able to reduce the number and growth of liver metastases and that it

was able to markedly prolong the survival of animals with liver metastases, is very promising. If phase I trial of Minnelide yields encouraging results, it could open doors for evaluation of

this novel compound in metastatic as well as primary colon cancer. The mechanism of action of triptolide or Minnelide is not clearly understood. Our previous studies show that triptolide

can induce cell death in cancer cells by multiple mechanisms. We have shown that, depending on the cell type, triptolide can induce either autophagy or apoptosis.15 In the current study we

demonstrate that triptolide induces apoptosis in colon cancer cells as well. Intriguingly, at higher concentrations the extent of apoptosis decreases as suggested by decreased caspase

activation and decreased annexin positivity. However, even with decreasing apoptosis, higher doses of triptolide were able to decrease the viability of colon cancer cell lines, suggesting

that another mechanism of cell death was functional at higher doses. Our data suggests that higher concentrations of triptolide did not induce autophagy or necrosis but did induce G1 cell

cycle arrest. Triptolide-induced cell cycle arrest has been seen in other cancer cell types including colon cancer.11, 18, 19 Zhao _et al_19 have shown that triptolide decreases

proliferation of multiple myeloma cells and induces G0–G1 cell cycle arrest. Zhang _et al_18 demonstrated that triptolide inhibits proliferation and invasion potential of malignant glioma

cell lines by inducing G0/G1 arrest. Similarly Liu _et al_11 have observed that triptolide induces G0–G1 cell cycle arrest without inducing apoptosis in colon cancer cell lines. That

triptolide induces a biphasic response, predominant apoptosis at low concentrations and cell cycle arrest at high concentrations, has not been described before. Various disease or cell

type-specific mechanisms of triptolide-induced G0/G1 arrest have been proposed. G1 to S transition is controlled by Rb, a member of the family of pocket proteins, and is mediated by its

binding to E2F1. The ability of Rb to bind to E2F1 and modulate its activity is in itself regulated by its cell cycle-dependent phosphorylation. Hypophosphorylated form of Rb predominates

during G0 and early G1 and binds to E2F. Increasing degree of Rb phosphorylation initially by CDK-4 and CDK-6 along with cyclin D and subsequently by CDK-2 in combination with cyclin E

causes E2F release and G1–S progression.22 Rb phosphorylation by cyclin/CDK complexes is further regulated by CDK inhibitors, the Ink4 protein family (p15, p16, p18, p19) and Cip/Kip protein

family (p21, p27, and p57). Cellular stresses as well as other inhibitors like transforming growth factor β can stall cell cycle progression through inhibition of cyclin/CDK complexes by

CDK inhibitors and maintenance of pRb in the hypophosphorylated form.23 Modulation of any of these events/proteins can lead to G1–S arrest. Previously published literature suggests that

triptolide could induce cell cycle arrest by modulating many of these proteins. Zhang _et al_18 have shown that triptolide induces G1–S arrest in glioblastoma cells by decreasing levels of

cyclin D1, CDK-4, and CDK-6 and by decreased phosphorylation of Rb. In prostate cancer, Li _et al_20 have demonstrated that that triptolide interferes with G1/S phase transition leading to a

loss of cell viability in two cell lines independent of their p53 status. Recently, a study by Liu _et al_11 in colon cancer cell lines HT29 and SW480 demonstrated that triptolide-induced

G1 arrest in these cell lines by increasing levels of p21, which then inhibits CDK-4 and CDK-6. This is intriguing as HT29 is a p53-mutated cell line and p21 is a p53-controlled gene.

Moreover, in this study the concentration of triptolide, which induces cell cycle arrest was lower (50 nM and lower) than what we observed in our study (50 nM and higher). In contrast, our

data suggest that triptolide induces cell cycle arrest in HCT116 (wild type p53) and HT29 (p53 mutant) by inhibiting E2F transactivation. Our data also suggest that triptolide does not

modulate Rb-E2F1 binding and does not decrease cyclin D or cyclin E, CDK-4 or CDK-6, or increase p21 levels. Results from chromatin immunoprecipitation assay demonstrate that

triptolide-mediated inhibition of E2F1 activity is not through inhibition of E2F1 binding to promoter sites. These together suggest that the inhibition of E2F1 transactivation must happen by

its effect on events beyond E2F1 DNA binding. Events that regulate E2F1 activity beyond its binding to DNA are not well known. Similar to many other transcription factors, E2F1

transcriptional activity has been shown to be regulated by acetylation.24 Also, triptolide has been shown to regulate the transcriptional activity of many other transcription factors like

NFκB25 and HSF-126 by influencing post-DNA-binding events. Elucidation of the post E2F1 DNA-binding events, which are modulated by triptolide would lead to progress in our understanding of

mechanism of action of triptolide. The fact that triptolide leads to apoptosis at low doses and cell cycle arrest at high doses is also very intriguing. Cell cycle arrest and apoptosis are

closely linked events and many of the events in apoptosis are regulated by genes involved in cell cycle progression.27 For example, c-Myc can induce both proliferation and apoptosis and cell

fate may depend on the presence or absence of mitogens.27 Similarly, p53 activation can induce cell cycle arrest or cell death depending on many factors including p21 status of cell,

crosstalk with pRb, and extent of DNA damage. Interestingly, data suggest that E2F besides regulating cell cycle regulates apoptotic pathways as well. Few of the apoptotic E2F1 target genes

include apoptosis protease-activating factor-1, p73, and ARF.28, 29 E2F1 can induce apoptosis in both p53-dependent and -independent fashion.22 It is interesting that in the current study

triptolide decreased E2F1 activity and at the same time a reduction in apoptotic parameters was observed. Though not conclusive, this may suggest that E2F1 inhibition may contribute to cell

cycle arrest and inhibition of apoptosis observed with high doses of triptolide. In summary, our data demonstrates that triptolide and its novel water soluble analog Minnelide are very

effective in killing colon cancer cells _in vitro_ and in reducing growth of primary colon tumors as well as colorectal metastases to the liver _in vivo,_ respectively. We also show that

triptolide leads to predominant apoptosis at low dose and cell cycle arrest at high dose. This is first report that reveals biphasic effect of triptolide on apoptosis and cell cycle. Our

results also suggest that triptolide induces cell cycle arrest by inhibiting E2F1 activity by inhibiting its transcriptional activity. Overall Minnelide could emerge as novel therapeutic

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Partnership for Biotechnology and Medical Genomics. AUTHOR INFORMATION Author notes * Amanda R Oliveira and Georg Beyer: These authors contributed equally to this work. AUTHORS AND

AFFILIATIONS * Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN, USA Amanda R Oliveira, Georg Beyer, Rohit Chugh, Steven J Skube,

 Kaustav Majumder, Sulagna Banerjee, Veena Sangwan, Lihua Li, Rajinder K Dawra, Subbaya Subramanian, Ashok K Saluja & Vikas Dudeja Authors * Amanda R Oliveira View author publications

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this author inPubMed Google Scholar * Ashok K Saluja View author publications You can also search for this author inPubMed Google Scholar * Vikas Dudeja View author publications You can also

search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Vikas Dudeja. ETHICS DECLARATIONS COMPETING INTERESTS R Chugh has ownership interest in a patent of

Minnelide. AK Saluja has ownership interest (including patents) in Minneamrita therapeutics and is a consultant/advisory board member for Minneamrita Therapeutics. The remaining authors

declare no conflict ofinterest. ADDITIONAL INFORMATION Supplementary Information accompanies the paper on the Laboratory Investigation website In this paper, the authors demonstrate that

minnelide, a water-soluble analog of triptolide, is effective against colon cancer in multiple animal models of colon cancer. Mechanistically, they show that at low concentrations,

triptolide induces apoptotic cell death but at higher concentrations it induces G1 cell cycle arrest by inhibiting transcriptional activation of E2F1 through modulation of events downstream

of DNA binding. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 (JPG 536 KB) SUPPLEMENTARY FIGURE 2 (JPG 629 KB) SUPPLEMENTARY FIGURE 3 (JPG 289 KB) SUPPLEMENTARY FIGURE 4 (JPG 191 KB)

SUPPLEMENTARY FIGURE LEGENDS (DOC 22 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Oliveira, A., Beyer, G., Chugh, R. _et al._ Triptolide abrogates

growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F. _Lab Invest_ 95, 648–659 (2015). https://doi.org/10.1038/labinvest.2015.46 Download

citation * Received: 19 August 2014 * Revised: 18 February 2015 * Accepted: 27 February 2015 * Published: 20 April 2015 * Issue Date: June 2015 * DOI:

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