Activity In Cervical Cancer Research Articles

Evaluation of anticancer activity of Plumbago indica root extract on cervical HeLa cancer cells

Plumbago indica L. has a various pharmacological activity; however, the anticancer activity in cervical cancer has less information. To examine the antiproliferative, apoptotic, and antimigratory activity of Plumbago indica root extract on cervical cancer cells. The extract of P. indica was explored for cell viability, apoptosis, and migration by sulforhodamine B, colony formation, cell cycle distribution, Wound healing, Annexin V-FITC staining apoptotic, mitochondrial function, and reactive oxygen species (ROS) formation. The data indicated that P. indica extract suppressed HeLa cervical cells viability by dose- and time-dependent manner along with the reduction of cancer cell number and colony forming ability. After 48 h of incubation period, the PR extract exhibited a significant reduction of migratory ability by dose-dependent manner. Furthermore, these extracts significantly showed a reduction of cell viability along with induction of late apoptosis. The mechanism of action was shown by detecting mitochondrial dysfunction and activating of ROS formation on HeLa cells-treated with P. indica extract. The extract of P. indica has a growth inhibition, migratory suppression, and apoptotic induction. The extract has an anticancer activity by reducing mitochondrial function and activating ROS formation.

CREB1 activation promotes human papillomavirus oncogene expression and cervical cancer cell transformation.

Human papillomaviruses (HPVs) infect the oral and anogenital mucosa and can cause cancer. The high-risk (HR)-HPV oncoproteins, E6 and E7, hijack cellular factors to promote cell proliferation, delay differentiation and induce genomic instability, thus predisposing infected cells to malignant transformation. cAMP response element (CRE)-binding protein 1 (CREB1) is a master transcription factor that can function as a proto-oncogene, the abnormal activity of which is associated with multiple cancers. However, little is known about the interplay between HPV and CREB1 activity in cervical cancer or the productive HPV lifecycle. We show that CREB is activated in productively infected primary keratinocytes and that CREB1 expression and phosphorylation is associated with the progression of HPV+ cervical disease. The depletion of CREB1 or inhibition of CREB1 activity results in decreased cell proliferation and reduced expression of markers of epithelial to mesenchymal transition, coupled with reduced migration in HPV+ cervical cancer cell lines. CREB1 expression is negatively regulated by the tumor suppressor microRNA, miR-203a, and CREB1 phosphorylation is controlled through the MAPK/MSK pathway. Crucially, CREB1 directly binds the viral promoter to upregulate transcription of the E6/E7 oncogenes, establishing a positive feedback loop between the HPV oncoproteins and CREB1. Our findings demonstrate the oncogenic function of CREB1 in HPV+ cervical cancer and its relationship with the HPV oncogenes.

Design, Synthesis, and Anti-Cervical Cancer and Reversal of Tumor Multidrug Resistance Activity of Novel Nitrogen-Containing Heterocyclic Chalcone Derivatives.

This study involved the design and synthesis of 21 new nitrogen-containing heterocyclic chalcone derivatives utilizing the active substructure splicing principle, with glycyrrhiza chalcone serving as the lead compound. The targets of these derivatives were VEGFR-2 and P-gp, and their efficacy against cervical cancer was evaluated. Following preliminary conformational analysis, compound 6f ((E)-1-(2-hydroxy-5-((4-hydroxypiperidin-1-yl)methyl)-4-methoxyphenyl)-3-(4-((4-methylpiperidin-1-yl)methyl)phenyl)prop-2-en-1-one) exhibited significant antiproliferative activity against human cervical cancer cells (HeLa and SiHa) with IC50 values of 6.52 ± 0.42 and 7.88 ± 0.52 μM, respectively, when compared to other compounds and positive control drugs. Additionally, this compound demonstrated lower toxicity towards human normal cervical epithelial cells (H8). Subsequent investigations have demonstrated that 6f exerts an inhibitory impact on VEGFR-2, as evidenced by its ability to impede the phosphorylation of p-VEGFR-2, p-PI3K, and p-Akt proteins in HeLa cells. This, in turn, results in the suppression of cell proliferation and the induction of both early and late apoptosis in a concentration-dependent manner. Furthermore, 6f significantly curtails the invasion and migration of HeLa cells. In addition, 6f had an IC50 of 7.74 ± 0.36 μM against human cervical cancer cisplatin-resistant HeLa/DDP cells and a resistance index (RI) of 1.19, compared to 7.36 for cisplatin HeLa cells. The combination of 6f and cisplatin resulted in a significant reduction in cisplatin resistance in HeLa/DDP cells. Molecular docking analyses revealed that 6f exhibited binding free energies of -9.074 and -9.823 kcal·mol-1 to VEGFR-2 and P-gp targets, respectively, and formed hydrogen bonding forces. These findings suggest that 6f has potential as an anti-cervical cancer agent and may reverse cisplatin-resistant activity in cervical cancer. The introduction of the 4-hydroxy piperidine and 4-methyl piperidine rings may contribute to its efficacy, and its mechanism of action may involve dual inhibition of VEGFR-2 and P-gp targets.

Blocking TIGIT/CD155 signalling reverses CD8+ T cell exhaustion and enhances the antitumor activity in cervical cancer

ObjectiveTIGIT/CD155 has attracted widespread attention as a new immune checkpoint and a potential target for cancer immunotherapy. In our study, we evaluated the role of TIGIT/CD155 checkpoints in the progression of cervical cancer.MethodsThe expression of CD155 and TIGIT in cervical cancer tissues was detected using flow cytometry, immunohistochemistry (IHC) and gene expression profiling. In vivo and in vitro experiments have proven that blocking TIGIT/CD155 restores the ability of CD8+ T cells to produce cytokines. Changes in the NF-κB and ERK pathways were detected using western blotting (WB) after blocking TIGIT/CD155 signalling.ResultsTIGIT expression was elevated in patients with cervical cancer. High TIGIT expression in CD8+ T lymphocytes from patients with cervical cancer promotes the exhaustion of CD8+ T lymphocytes. In addition, CD155 is expressed at high levels in cervical cancer tissues and is negatively correlated with the level of infiltrating CD8+ T cells. We found that TIGIT, upon binding to CD155 and being phosphorylated, inhibited NF-κB and ERK activation by recruiting SHIP-1, resulting in the downregulation of cytokine production. Blocking TIGIT in activated CD8+ T cells attenuates the inhibitory effect of SHIP-1 on CD8+ T cells and enhances the activation of NF-κB and ERK. In vivo and in vitro experiments have proven that blocking TIGIT/CD155 restores the ability of CD8+ T cells to produce cytokines. Injecting the blocking antibody TIGIT in vivo inhibits tumour growth and enhances CD8+ T lymphocyte function. Treatment with a combination of TIGIT and PD-1 inhibitors further increases the efficacy of the TIGIT blocking antibody.ConclusionsOur research shows that TIGIT/CD155 is a potential therapeutic target for cervical cancer.

Trial in progress update on ENGOT-cx8/GOG-3024/innovaTV 205: Addition of a new cohort with first-line (1L) tisotumab vedotin (TV) + pembrolizumab (pembro) + carboplatin (carbo) ± bevacizumab (bev) in recurrent/metastatic cervical cancer (r/mCC).

TPS5603 Background: Despite the use of platinum-taxane doublets ± bev in eligible patients (pts), overall survival (OS) outcomes for pts with r/mCC remain poor. With the US approval of pembro + chemotherapy ± bev in the 1L setting for r/mCC with ≥1% of programmed death ligand 1–positive cells in the tumor, an unmet need remains for pts who do not meet this threshold and for pts who progress or are intolerant to standard treatment (tx). TV is a tissue factor–directed antibody–drug conjugate that has been granted accelerated approval in the United States for the tx of adults with r/mCC with disease progression on or after chemotherapy. To develop more effective treatments, we investigated TV in combination with agents with known activity in cervical cancer. We conducted a 2-part, multicohort phase 1b/2 trial, ENGOT-cx8/GOG-3024/innovaTV 205 (NCT03786081), to evaluate TV in combination with bev, pembro, or carbo. The phase 1b dose-escalation phase of innovaTV 205 established the recommended phase 2 dose (RP2D) and the feasibility of these doublet combinations (Monk et al, IGCS 2021). Moreover, we recently reported encouraging antitumor activity from the dose-expansion cohort of TV + carbo (1L; confirmed objective response rate [ORR], 55%; median duration of response [DOR], 8.3 mo) and TV + pembro (second-line; confirmed ORR, 38%; median DOR, 13.8 mo) (Vergote et al, ESMO 2021). The current report describes the design of a new, ongoing dose expansion cohort in the innovaTV 205 study to evaluate the combinations of TV, pembro, and carbo ± bev. Methods: A new cohort has been added to the innovaTV 205 study, comprising adult pts with recurrent or stage IVB squamous, adenosquamous, or adenocarcinoma of the cervix with no prior systemic therapy and an Eastern Cooperative Oncology Group score of 0 or 1. Pts will be treated with the RP2D of TV (2.0 mg/kg) + carbo (AUC 5 mg/mL), pembro (200 mg), and bev (15 mg/kg) every 3 weeks or with TV + carbo (AUC 5 mg/mL) and pembro (200 mg). To assess the regimen’s initial tolerability, there will be a dose-limiting toxicity evaluation period that will consist of completion of 1 tx cycle of 21 days for 6 pts enrolled to receive the quadruplet combination. The primary endpoint of this dose expansion phase is confirmed ORR per RECIST v1.1; secondary endpoints include DOR, time to response, progression-free survival, OS, and safety. Enrollment is ongoing in the United States and Europe, with additional sites planned globally. Clinical trial information: NCT03786081.

Antitumor activity of pachymic acid in cervical cancer through inducing endoplasmic reticulum stress, mitochondrial dysfunction, and activating the AMPK pathway.

Pachymic acid has various pharmacological effects, including anti-inflammatory, antioxidant, immunomodulatory, and antitumor. However, the role of pachymic acid in cervical cancer remains unclear. So, we investigated the effects of pachymic acid in cervical cancer and elucidated the underlying mechanisms. We treated HeLa cells and normal cervical epithelial cells (HUCECs) with pachymic acid (0, 10, 20, 40, 80, or 160 μM) for 72 h, and found the cell activity was decreased in cells treated with 160 μM pachymic acid for 48 h or 80 μM pachymic acid for 72 h, while HUCECs viability without effect. Next, we observed that endoplasmic reticulum (ER) related gene expression, mitochondrial membrane potential (MMP) changes, ATP depletion, reactive oxygen species (ROS) generation and apoptosis were increased. Moreover, we observed that cytochrome C (Cytc) expression was increased and apoptosis-inducing factor (AIF) was decreased in the cytoplasm of pachymic acid-treated HeLa cells. Tauroursodeoxycholic acid (TUDCA) of ER stress inhibitor reversed the effects of pachymic acid on HeLa cells. Phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) of the AMPK pathway key protein was upregulated in pachymic acid-induced HeLa cells. Finally, we subcutaneously implanted HeLa cells into female nude mice and treated them with pachymic acid (50 mg/kg) for 3 weeks (5 days/week), and observed in pachymic acid induced xenograft mice, tumor growth was suppressed, cell apoptosis, ER-related gene expression, and ROS levels in tumor tissues were increased. Therefore, these findings demonstrated that pachymic acid plays an anti-tumor activity in cervical cancer through inducing ER stress, mitochondrial dysfunction, and activating the AMPK pathway.

Matrine exerts antitumor activity in cervical cancer by protective autophagy via the Akt/mTOR pathway in vitro and in vivo.

Matrine is a quinazoline alkaloid extracted from Sophora flavescens. The aim of the present study was to determine whether matrine can induce autophagy in the human HeLa and SiHa cervical cancer cell lines in vitro and in vivo. Cell viability assay was used to assess the suppressive effect of matrine and cisplatin on the proliferation of HeLa and SiHa cells. A total of 28 4-week-old female BALB/c nude mice were used for the in vivo study. Autophagy and protein expression were observed via transmission electron microscopy, monodansylcadaverine and immunohistochemical staining and western blotting. The inhibitory effect of matrine on the proliferation of cervical cancer cells was time- and dose-dependent. The combination of matrine and cisplatin synergistically inhibited the proliferation of cervical cancer cells in vitro and in vivo. Transmission electron microscopy showed that after the addition of matrine, numerous autophagosomes and autophagolysosomes were observable in HeLa and SiHa cells, as demonstrated by monodansylcadaverine staining. Western blotting and immunohistochemical staining showed that as the concentration of matrine increased, the expression of the autophagy marker LC3A/B-II also increased significantly in vitro and in vivo. These findings suggested that matrine inhibited the proliferation of cervical cancer cells and induced autophagy by inhibiting the Akt/mTOR signaling pathway. Thus, matrine may represented a potential candidate in combination therapy for cervical cancer as an inducer of autophagy.

Investigating the Effects of RBBP6 Gene Expression on Telomerase Activity in Cervical Cancer Cells.

BackgroundRBBP6 is one of the genes identified as a proliferative gene that plays a role in cancer development, On the other hand both RBBP6 and telomerase activity have been shown to be increase in various cancers. E6 protein of HPV and RBBP6 is known to enhance the progression of cancer cells by interacting with p53 and presenting it to be ubiquitinated by the proteasome thereby promoting cell proliferation and preventing apoptosis. Studies also show that HPV E6 protein can increase telomerase activity by activating the expression of human telomerase reverse transcriptase (hTERT), thus enabling the immortalization of the cells. With RBBP6 and hTERT showing a similar profile in cancer cells, we seek to investigate any possible effect of RBBP6 on telomerase activity.ResultsUsing real-time qPCR and TRAPeze RT Telomerase detection kit (Merc) respectively. We used cervical cancer cell lines in which CaSki cell showed the reduction of hTERT expression and reduction in telomerase activity significantly in RBBP6-knockdown cells. While no significant changes were observed in HeLa cells. Real-time growth assay revealed a significant drop in cell growth in co-silenced RBBP6 and hTERT cells substantiating our observation that RBBP6 might be playing a role in cell proliferation.ConclusionTaken all together, the observed effect of RBBP6 gene silencing on telomerase activity in cervical cancer is cell line dependent.

Plantamajoside inhibits hypoxia-induced migration and invasion of human cervical cancer cells through the NF-κB and PI3K/akt pathways

Plantamajoside (PMS) is a major compound of Plantago asiatica and possesses anti-tumor property in several types of cancers. However, the effect of PMS on cervical cancer has not been investigated. This study aimed to investigate the effect of PMS on the migration and invasion of cervical cancer cell lines under hypoxic condition. Our results demonstrated that PMS significantly inhibited hypoxia-caused increases in cell migration and invasion of cervical cancer cells. The hypoxia-induced epithelial-mesenchymal transition (EMT) process was prevented by PMS with increased E-cadherin expression, and decreased expression levels of N-cadherin and vimentin in cervical cancer cells. Besides, the expression levels of transcription factors slug and snail were suppressed by PMS in hypoxia-induced cervical cancer cells. The increased mRNA and protein levels of hypoxia-inducible factor 1alpha (HIF-1α) in hypoxia-induced cervical cancer cells were prevented by PMS. Furthermore, PMS blocked the hypoxia-induced activation of NF-κB and PI3K/Akt pathway in cervical cancer cells. Taken together, these findings suggest that PMS exerted an anti-tumor activity in cervical cancer through preventing the hypoxia-induced EMT. Thus, PMS might serve as a therapeutic agent for the treatment of cervical cancer.

Advances in immunotherapy for cervical cancer.

Novel therapies are needed for the treatment of recurrent cervical cancer. The best chemotherapy regimen to date has a response rate of 48% with an overall survival of 17 months, with limited options for second-line chemotherapy. Immunotherapy can induce a strong immune response in cervical cancer due to retained viral antigens and is reviewed in this article. Current clinical trials include treatment with Listeria that elicits an immune response against the E7 oncoprotein and active vaccines against the E7 oncoprotein. Although the response rates to programmed cell death 1 (PD-1) inhibition alone have been modest, the landmark survival reported in these trials suggests the activity of these agents may not be measured by RECIST criteria. The KEYNOTE-158 trial has led to the approval of pembrolizumab in recurrent programmed cell death ligand 1 (PD-L1) positive cervical cancer. Combinations of programmed cell death 1 and anticytotoxic T-lymphocyte-associated protein 4 inhibitors (CTLA4) inhibitors have shown promising and durable activity. There is active research with new combinations of checkpoint inhibitors, as well as combinations of these drugs with chemotherapy and radiation, and other novel approaches. Immune therapy has broad activity in cervical cancer. Responses to immunotherapy can be dramatic and durable. Continued work to find the optimal combination and setting for immunotherapy is ongoing.

Eupatilin regulates proliferation and cell cycle of cervical cancer by regulating hedgehog signalling pathway

Eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone) is a natural active substance found in génépi group plants, and its pharmacological activities has been proven to be useful in the treatment of various cancers. However, whether eupatilin demonstrates anti-cancer activity in cervical cancer is still under evaluation. To clarify this, cancer cell lines and nude mouse model were used in this study. The results indicated that eupatilin could inhibit the occurrence of cervical cancer both in vivo and in vitro. Cervical cancer cell lines (C4-1, HeLa, Caski, and Siha) and Ect1/E6E7 cells were incubated with eupatilin (40μM) for 48 hours. Compared with the control group, the viability of cervical cancer cells decreased significantly, while the apoptotic cells increased significantly. Cell cycle analysis showed that eupatilin treatment of HeLa and Caski cells reduced the proliferation index. Eupatilin at 40 mg/kg also inhibited tumour growth in tumour-bearing mice. Interestingly, weakened hedgehog signalling was observed in cervical cancer cells and tumours from tumour-bearing mice after eupatilin treatment. Our results reveal the inhibitory effect of eupatilin on cervical cancer and shed new light on the molecular mechanism of its therapeutic effect. SIGNIFICANCE OF THE STUDY: Eupatilin inhibited proliferation via promoting apoptosis and cell cycle arrest in HeLa and Caski cervical cancer cell lines. In addition, nude mouse tumourigenicity assay proved that eupatilin can suppress tumour growth in vivo. Dramatically, these activities might be involved in hedgehog signal pathway.

Natural killer T cell cytotoxic activity in cervical cancer is facilitated by the LINC00240/microRNA-124-3p/STAT3/MICA axis

Long noncoding RNAs play significant roles in diverse cancers. In this study, we found that LINC00240 expression was markedly increased in cervical cancer. Functional in vitro assays in cervical cancer cells showed that LINC00240 enhanced the growth, migration, and invasion of cervical cancer cells. The target of LINC00240 was confirmed as microRNA(miR)-124-3p. Inhibition of miR-124-3p significantly enhanced cervical cancer progression via targeting of STAT3, which is greatly activated in tumor-infiltrating immune cells. LINC00240 expression was able to induce STAT3 expression via sponging of miR-124-3p, and showed a positive association with STAT3 expression in cervical cancer tissues. MHC class I-related chain (MIC)-A plays a key role in activating natural killer T (NKT) cells and serves as a downstream target of STAT3. Here, MICA was inhibited by up-regulation of LINC00240, and could be rescued by STAT3 knockdown. In addition, LINC00240 overexpression suppressed the cytotoxic activity of NKT cells by affecting the STAT3/MICA axis. Subsequently, we found that LINC00240 expression promoted cervical cancer progression via induction of miR-124-3p/STAT3/MICA-mediated NKT cell tolerance. Considering these findings, we conclude that LINC00240 might be a novel target for cervical cancer.

Piperlongumine increases the apoptotic effect of doxorubicin and paclitaxel in a cervical cancer cell line.

Piperlongumine (PL) is an alkaloid derived from the edible pepper (Piper longum L) and it has been described to have various biologic activities including anticancer effects. Our aim in this study was to assess the cytotoxic role of PL on a cervical cancer cell line (HeLa) and to evaluate the effects of PL/doxorubicin and PL/paclitaxel combination therapies on apoptotic cancer cell death. The cytotoxicity, IC50 doses by MTT assay confirmed by fluorescent imaging, and apoptotic cell rates by Annexin V staining using flow cytometry were determined for PL, doxorubicin, paclitaxel, and for their combinations. It was shown that the PL by itself induced the apoptosis in HeLa cells. PL in combination with doxorubicin and paclitaxel increased apoptotic cell death compared to either chemotherapeutic agent alone. We conclude that the PL inhibits cancer cell growth by inducing apoptosis and has a potential anticancer activity in cervical cancer, especially when combined with doxorubicin and paclitaxel.

Inhibiting MNK Selectively Targets Cervical Cancer via Suppressing eIF4E-Mediated β-Catenin Activation

Targeting β-catenin has been shown to have great potential therapeutic value in cervical cancer. Because β-catenin is also essential for normal cells, strategies to specifically target cancer will require identification of druggable factors capable of distinguishing β-catenin signaling pathways between cancer and normal cells. Expression of p-eIF4E and p-β-catenin was analyzed in malignant and normal cervical tissues and cells. The effects and its underlying mechanisms of targeting MNK and eukaryotic translation initiation factor 4E (eIF4E) were determined in cervical cancer and normal cells. Inhibiting MNK/eIF4E axis selectively targets cervical cancer without affecting normal cervical cells, via suppressing eIF4E-mediated β-catenin activation. We found that eIF4E phosphorylation was upregulated in cervical cancer cells and tissues but not normal cervical counterparts, and its phosphorylation at Ser 209 activates Wnt/β-catenin signaling, promotes growth and migration in cervical cancer, in an MNK-dependent manner. MNK inhibition via genetic small interfering RNA (siRNA) knockdown or pharmacologic inhibitor effectively decreased phosphorylation of eIF4E and β-catenin, leading to reduced β-catenin activity and transcript levels of Wnt target genes in cervical cancer cells. Consistently, we found that MNK kinase inhibitor is effective in inhibiting proliferation and migration, and inducing apoptosis in cervical cancer but not normal cervical cells. The combination of MNK kinase inhibitor with paclitaxel achieved greater efficacy in cervical cancer cells than paclitaxel alone. Our work identifies MNK-eIF4E axis as a specific and critical regulator of β-catenin activity in cervical cancer but not normal cervical cells, and suggests that targeting MNK is a useful therapeutic strategy in cervical cancer.

CDK7 inhibitor suppresses tumor progression through blocking the cell cycle at the G2/M phase and inhibiting transcriptional activity in cervical cancer.

BackgroundThe disordered cell cycle and dysregulated expression of numerous oncogenes involved in tumor-relevant processes are highly related to the tumorigenesis of cervical cancer. Cyclin-dependent kinase 7 (CDK7) constitutes the indispensable catalytic subunit of CDK-activating kinase (CAK), which is required for both cell cycle transition and transcriptional regulation. However, research regarding the antitumor effects of CDK7 inhibition in cervical cancer remains unclear.PurposeOur study aims to explore the antineoplastic effects of the CDK7 inhibitor THZ1 in cervical cancer cells and to find a potential agent for cervical cancer treatment.MethodsThe CRISPR-Cas9 system was used to knock down CDK7. The Cell Counting Kit-8 (CCK-8) assay was used to detect the cell viability after CDK7 depletion and THZ1 treatment. Western blot was employed to detect protein expression. The expression levels of mRNA were assayed through qRT-PCR. Flow cytometry analysis was used to assay the apoptotic cells and cell cycle distribution. Gene expression microarray analysis was used to identify the differential expression of the genes. Subcutaneous xenograft mouse model was performed to test the antineoplastic effects of THZ1 in vivo.ResultsWe revealed that the genetic depletion of CDK7 using the CRISPR-Cas9 system exhibited great cell growth inhibition in cervical cancer cell lines, consistent with the effects of CDK7 blocking using THZ1. Cervical cancer cells were highly sensitive to THZ1 treatment, and a low concentration of THZ1 could induce substantial cell apoptosis. THZ1 specifically perturbed the phosphorylation of cell cycle regulator CDK1 and decreased the expression of cyclin B1, leading to a cell cycle blockage at the G2/M phase and inducing cell growth inhibition. The gene expression microarray analysis showed that massive oncogene transcripts, especially those associated with tumorigenesis, were preferential suppressed after THZ1 treatment. The qRT-PCR confirmed that several essential oncogenes in tumorigenesis (c-MYC, hTERT, RAD51, and BCL-2) and HPV viral oncogenes (E6 and E7) were preferentially repressed by THZ1. Moreover, THZ1 exhibited substantial antineoplastic effects against cervical cancer in vivo without inducing obvious side effects.ConclusionThese findings indicated that the CDK7 inhibitor THZ1 is a potential option in cervical cancer treatment owing to its ability to inhibit cell cycle progression and transcriptional activity.

Dicerandrol B: a natural xanthone dimer induces apoptosis in cervical cancer HeLa cells through the endoplasmic reticulum stress and mitochondrial damage.

BackgroundDicerandrol B is a natural antitumor agent that can be isolated from the endophytic fungus, Phomopsis sp. The present study investigated the effects of dicerandrol B on human cervical cancer HeLa cells.Materials and methodsIn this study, dicerandrol B was identified by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We used MTT to detect the cell viability. Flow cytometry was used to analyze the apoptosis and cell cycle. Western blot was used to examine the expression of related proteins.ResultsDicerandrol B was isolated from the endophytic fungus Phomopsis sp. The MTT assay and flow cytometry showed that dicerandrol B significantly inhibited HeLa cell viability and induced G2/M cell cycle arrest. Western blot analysis demonstrated that dicerandrol B increased the levels of GRP78, ubiquitin, cleaved PARP, and Bax protein, decreased the levels of PARP and Bcl-2 protein, and caused an increase in the Bax/Bcl-2 ratio in HeLa cells. Dicerandrol B increased the production of ROS in HeLa cells, which was attenuated by the antioxidant N-acetyl-l-cysteine.ConclusionThese findings suggest that dicerandrol B induces apoptosis in human HeLa cells, possibly through the endoplasmic reticulum stress and mitochondrial apoptotic pathways. This suggests that dicerandrol B possesses strong anticancer activity in cervical cancer and provides insight into the underlying mechanisms.

Cucurbitacin D exhibits potent anti-cancer activity in cervical cancer.

In this study, we for the first time, investigated the potential anti-cancer effects of a novel analogue of cucurbitacin (Cucurbitacin D) against cervical cancer in vitro and in vivo. Cucurbitacin D inhibited viability and growth of cervical cancer cells (CaSki and SiHa) in a dose-dependent manner. IC50 of Cucurbitacin D was recorded at 400 nM and 250 nM in CaSki and SiHa cells, respectively. Induction of apoptosis was observed in Cucurbitacin D treated cervical cancer cells as measured by enhanced Annexin V staining and cleavage in PARP protein. Cucurbitacin D treatment of cervical cancer cells arrested the cell cycle in G1/S phase, inhibited constitutive expression of E6, Cyclin D1, CDK4, pRb, and Rb and induced the protein levels of p21 and p27. Cucurbitacin D also inhibited phosphorylation of STAT3 at Ser727 and Tyr705 residues as well as its downstream target genes c-Myc, and MMP9. Cucurbitacin D enhanced the expression of tumor suppressor microRNAs (miR-145, miRNA-143, and miRNA34a) in cervical cancer cells. Cucurbitacin D treatment (1 mg/kg body weight) effectively inhibited growth of cervical cancer cells derived orthotopic xenograft tumors in athymic nude mice. These results demonstrate the potential therapeutic efficacy of Cucurbitacin D against cervical cancer.

Abstract 3081: Novel cucurbitacin analogue Cuc D exhibits potent anti-cancer activity in cervical cancer

Abstract Background: Cervical cancer is one of the leading cause of mortality among women in US. Naturally occurring dietary compounds have gained increasing attention for their anticancer effects. Cucurbitacins, tetracyclic triterpenoid compound, belong to a family of Cucurbitaceae have shown promising anti-cancer activity. Herein, we investigated the potential anti-cancer effects of a novel analogue of cucurbitacin D (Cuc D) against cervical cancer in in vitro and in a xenograft mouse model. Methods: In our study, we used human cervical cancer cells (CaSki and SiHa). Cells were treated with Cuc D (0.05 to 1μM) for 48 and 72 hrs. MTS and colony formation assays were performed to investigate the effects of Cuc D on cell viability and proliferation. Western Blot analysis was performed to investigate the effects of Cuc D on cell proliferation and apoptotic markers. To determine the therapeutic effects of Cuc D, we used female athymic nude mice and injected CaSki cells (4 × 106) into the cervix to develop orthotopic xenograft tumors. Cuc D (1 mg/kg body weight) was administered through intratumoral injection four weeks post-tumor cell injection. Tumor volume in these mice were recorded bi-weekly. Results: Cuc D inhibited cell viability of cervical cancer cells in a dose-dependent manner. IC50 of Cuc D was observed 400 nM and 250 nM in Caski and SiHa cells, respectively. Cuc D treatment effectively inhibited growth of cervical cancer cells which was determined by decreased cell proliferation and colony formation assays. Cuc D treatment induced apoptosis in cervical cancer cells as measured by enhanced Annexin V staining. Western blot result also illustrated cleavage in PARP protein in Cuc D treated cells which further confirms apoptosis induction. Cuc D treatment also inhibited PI3K and c-Myc protein levels and phosphorylation of STAT3 and Rb proteins. In addition, Cuc D treatment induced the cell cycle inhibitory proteins (p21 and p27) and PTEN and the expression of a tumor suppressor microRNA, miR-145, as determined by qRT-PCR. In an orthotopic tumor xenograft mouse model, Cuc D treatment effectively inhibited tumor growth as compared to vehicle control treated mice. Conclusion: Taken together, our results demonstrate potent anti-cancer efficacy of Cuc D in cervical cancer cells via modulation of key onco/tumor suppressor proteins. Thus, Cuc D could be a useful therapeutic agent for cervical cancer treatment. Citation Format: Mohammed Sikander, Bilal Bin Hafeez, Fathi T. Halaweish, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Novel cucurbitacin analogue Cuc D exhibits potent anti-cancer activity in cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3081.

Abstract LB-095: HPV E7 antigen-expressing Listeria-based immunotherapy (ADXS11-001) prior to robotic surgery for HPV-positive oropharyngeal cancer enhances HPV-specific T cell immunity

Abstract Human papilloma virus-associated oropharyngeal cancer (HPVOPC), which accounts for almost 75% of newly diagnosed OPC, is an appealing target for immunotherapy due to the expression of viral antigens. ADXS11-001, a live attenuated Listeria monocytogenes listeriolysin O (LLO) immunotherapeutic agent expressing an HPV16-E7 fusion protein, has been shown to induce HPV-specific T cell responses in animal models, and to have clinical activity in cervical cancer. A phase II “window of opportunity” trial was designed to evaluate the effect of ADXS11-001 on anti-tumor immunity in peripheral blood and the tumor immune microenvironment (TIME) of patients with HPVOPC. Previously untreated, surgically resectable, stage II-IV, HPVOPC patients received two doses of ADXS11-001 over 5 weeks prior to transoral robotic surgery. Formalin-fixed paraffin embedded (FFPE) pre-treatment biopsies and post-treatment surgical resection specimens were banked for studies of the TIME. Peripheral blood samples were collected at multiple time points before, during and after ADXS11-001 administration and after surgery. The TIME was profiled by quantitative multiplex immunofluorescence (qIF) microscopy and conventional H&E histopathology. T cell immune responses in the peripheral blood were monitored by ELISPOT of IFN-γ and TNF-α expressing T cells. Serum expression of 38 cytokines was measured using the Luminex multiplex analysis platform. So far 8 patients have completed treatment, of which 5 showed increased E6 or E7-specific IFN-γ responses on the day of surgery or 5 weeks post-surgery, compared to pre-treatment responses. Serum cytokines CCL22 and CXCL10 showed a trend towards increase after ADXS11-001 delivery (p = 0.067, p = 0.070) and drop following surgery (p = 0.069, p = 0.016). CCL22 levels were correlated with E6-specific T cell response after immunotherapy treatment (R2 = 0.6303, p = 0.019). qIF results demonstrated increased post-treatment CD8 and CD4 intratumoral T cell infiltration in 4/8 patients, and correlations between intra-tumoral pre- and post-treatment CD8 numbers (R2 = 0.6481, p = 0.0167). Also, post-treatment PD-1 expression strongly correlated with PD-L1 expression (R2 = 0.8803, p = 0.0007) in the tumor. Tumor infiltrating lymphocytes at the tumor host interface tended to increase after treatment (p = 0.188) and correlated with pre-treatment numbers (R2 = 0.772, p = 0.042). Antigen-specific peripheral blood T cell responses are increased post-treatment with ADXS11-001 and correlate with increased serum CCL22 levels. In this small patient sample (n = 8), the intratumoral qIF and H&E results present an overall suggestion of positive treatment-induced effects in the TIME, which must be confirmed as additional patients are accrued. Citation Format: Rosemarie Krupar, Naoko Imai, Brett Miles, Eric Genden, Krzys Misiukiewicz, Yvonne Saenger, Elizabeth G. Demicco, Jigneshkumar Patel, Phapichaya Chaoprang Herrera, Falguni Parikh, Michael Donovan, Seunghee Kim-Schulze, Marshall Posner, Sacha Gnjatic, Andrew G. Sikora. HPV E7 antigen-expressing Listeria-based immunotherapy (ADXS11-001) prior to robotic surgery for HPV-positive oropharyngeal cancer enhances HPV-specific T cell immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-095.

Abstract 2536: AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells

Abstract Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers. It is also an indispensable factor for efficient expression of high risk HPV oncogenes E6/E7. Recent studies demonstrated the role of AP-1 in promoting stemness/quiescence of cancer stem cells. We have recently reported that HPVE6 oncogene controls stemness and self-renewal of cervical cancer stem cells through Hes1/NOTCH-1expression but little is known about factor(s) responsible for chemo- radioresistance of cancer stem cells. To understand this we have dissected the role of AP-1and its family members by using a herbal compound, curcumin and UV irradiation in cervical cancer. We isolated cervical cancer stem cells and enriched them by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133). The isolated cervical cancer stem cells (SP+ve→CD49+veCD71−ve→CD133+ve) designated as CaCxSLCs displayed spheroid forming and self-renewal property with increased expression of HPVE6, AP-1 transcripts and a higher AP-1 DNA-binding activity compared to non-stem cancer cells (NSP−ve→CD49−veCD71+ve→ CD133−ve) or unsorted parental cells. Treatment with UV-radiation alone resulted in clonogenic survival of CaCxSLCs but not non-stem cancer cells with enhanced AP-1 expression and activation. In contrast, CaCxSLCs treated with UV in combination with curcumin, resulted in complete loss of AP-1 DNA-binding activity followed by decrease in sphere formation and percentage of SP cells but an increased expression of the fos-related antigen-1 (Fra-1) which has been earlier shown to us have tumor suppressor activity in cervical cancer. Our study demonstrates a critical role of AP-1/Fra-1 signaling in imparting radiosensitization of cervical cancer stem cells. Curcumin, which down regulates almost all signalling pathways, including all members of AP-1 except Fra-1 indicating its role in chemo-radiosensitization of cancer stem cells. Thus combining curcumin with conventional cancer drugs may make cancer treatment most effective. Citation Format: Bhudev C. Das, Abhishek Tyagi, Bal Gangadhar Roy, Alok C. Bharti. AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2536.