ID: 1040 Anticancer effects of natural products from animal and plant origin

For last couple of decades, natural products have served us well in combating different types of cancer. The main sources of these useful compounds are from both animal and plant origin. Here we will present anticancer ability of bee venom (BV) and apigenin (API) towards different types of cancer cells in vitro. BV from honey bees is a complex mixture of a variety of different active peptides while API is a natural flavonoid found in several dietary plant foods. Anticancer effect of whole BV was tested in human cervical carcinoma HeLa cells and their drug-resistant HeLa CK subline while anticancer effect of API was tested in human breast cancer MCF-7 and MDA MB-231 cells. Cytotoxicity of both compounds towards cancer cells was evaluated by MTT assay whereas type of cell death was analysed by differential staining using acridine orange/ethidium bromide and was further verified by Western blot analysis. BV displayed dose-dependent cytotoxicity against both cell lines tested with drug-resistant HeLa CK cells being more sensitive to BV than their parental cell lines. Similarly, API inhibited the growth of both cell lines in a dose-dependent manner with MCF-7 cells being more sensitive. Treatment with BV induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium bromide and a lack of cleavage of apoptotic marker poly (ADP-ribose) polymerase (PARP) on Western blot. On the contrary, cell treated with API showed apoptosis as a dominant type of cell death in both cell lines which was further verified by Western blot analysis detecting cleaved PARP. In view of accumulating evidence on anti-proliferative and pro-cell death activity, both tested compounds could be used in the development of future anticancer drugs. Undoubtedly, therapeutic applications of BV and API are promising, however further in vitro and in vivo studies are warranted to resolve precise mechanisms responsible for their anticancer properties

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The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides symington type SS fruit include effects on mode of cell death

Pharmacognosy Magazine ◽

10.4103/0973-1296.157718 ◽

2015 ◽

Vol 11 (42) ◽

pp. 148 ◽

Cited By ~ 4

Author(s):

Theera Srisawat ◽

Yaowapa Sukpondma ◽

Potchanapond Graidist ◽

Siriphon Chimplee ◽

Kanyanatt Kanokwiroon

Keyword(s):

Cell Death ◽

Cell Lines ◽

Dose Dependent ◽

Mcf 7

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The In Vitro Anticancer Activity and Potential Mechanism of Action of 1-[(1R,2S)-2-fluorocyclopropyl]Ciprofloxacin-(4-methyl/phenyl/benzyl-3- aryl)-1,2,4-triazole-5(4H)-thione Hybrids

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200310123723 ◽

2020 ◽

Vol 20 (16) ◽

pp. 1493-1498

Author(s):

Ya-Zhou Zhang ◽

Hai-Lin Liu ◽

Qian-Song He ◽

Zhi Xu

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Anticancer Agents ◽

Topoisomerase Ii ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Drug Resistant ◽

Mcf 7

Aims: Development of 1-[(1R, 2S)-2-fluorocyclopropyl]ciprofloxacin-1,2,4-triazole-5(4H)- thione hybrids as potential dual-acting mechanism anticancer agent to overcome the drug resistance. Background: Chemotherapy is an essential tool for the treatment of lung and female breast cancers, and numerous anticancer agents have been launched for this purpose. However, the clinical outcomes of chemotherapy are usually far from satisfactory due to the side effects and resistance to chemotherapeutic drugs. Thus, it is urgent to develop novel anti-lung and anti-breast cancer agents. Background: Chemotherapy is an essential tool for the treatment of lung and female breast cancers, and numerous anticancer agents have been launched for this purpose. However, the clinical outcomes of chemotherapy are usually far from satisfactory due to the side effects and resistance to chemotherapeutic drugs. Thus, it is urgent to develop novel anti-lung and anti-breast cancer agents. Objective: The primary objective of this study was to evaluate the potential of bis-isatin scaffolds with alkyl/ether linkers between the two isatin moieties against different human breast cancer cell lines including A549, MCF-7 and their drug-resistant counterparts A549/CDDP, MCF-7/ADM cells. Methods: The 1-[(1R, 2S)-2-fluorocyclopropyl]ciprofloxacin-(4-methyl/phenyl/benzyl-3-aryl)-1,2,4- triazole-5(4H)-thione hybrids were screened for their in vitro activity against drug-sensitive lung (A549), breast (MCF-7) and their drug-resistant counterparts A549/CDDP (cisplatin-resistant), MCF- 7/ADM (doxorubicin-resistant) cancer cell lines by MTT assay. The inhibitory activity of these hybrids against topoisomerase II and EGFR was also evaluated to investigate the potential mechanism of action of these hybrids. Result: The most prominent hybrid 7k (IC50: 37.28-49.05 µM) was comparable to Vorinostat against A549 and A549/CDDP lung cancer cells, and was 2.79-2.94 times more active than Vorinostat against MCF-7 and MCF-7/ADM breast cancer cell lines. Moreover, hybrid 7k (IC50: 8.6 and 16.4 µM) also demonstrated dual inhibition against topoisomerase II and EGFR. Conclusion: The 1-[(1R, 2S)-2-fluorocyclopropyl]ciprofloxacin-1,2,4-triazole-5(4H)-thione hybrids possess equally activity against both drug-sensitive cancer cells and their drug-resistant counterparts, and the majority of them were no inferior to the reference Vorinostat. The mechanistic study revealed that these hybrids could inhibit both topoisomerase II and EGFR, so these hybrids can be developed as dual-acting mechanism anticancer agents.

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Novel CRM-1 Inhibitors for Therapy In Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.2734.2734 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2734-2734

Author(s):

Kejie Zhang ◽

Lan V Pham ◽

Liang Zhang ◽

Archito T. Tamayo ◽

Zhishuo Ou ◽

...

Keyword(s):

B Cells ◽

Western Blot ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Parp Cleavage ◽

Mantle Cell ◽

Dose Dependent

Abstract Abstract 2734 Chromosomal Region Maintenance 1 (CRM1) overexpression has been associated with cancer progression and mortality in several human cancers, suggesting that activation of nuclear export may play a role in human neoplasia and may serve as a novel target for the treatment of cancers. This overexpression of CRM1 may be related to the export of most tumor suppressor and growth regulatory proteins out of the nucleus, thereby functionally inactivating them. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that is not yet curable. The objective of our study was to investigate the status of CRM1 in MCL, both in MCL cell lines and primary MCL cells, in comparison to normal B cells, and to evaluate the therapeutic efficiency of CRM1 inhibition in MCL in vitro and in vivo, and to elucidate the mechanism of CRM1 inhibitor-mediated MCL cell apoptosis. We used 8 established MCL cell lines and primary cells from 4 patients with relapsed/refractory MCL. KPT185 and KPT276 are novel, highly selective, drug-like small molecular CRM1 inhibitors. Western Blot analysis showed that CRM1 was expressed in both the cytoplasm and nuclei of 8 MCL cell lines. CRM1 was mainly detected in nuclei of normal resting B cells; In contrast, CRM1 was primarily detected in the cytoplasm of freshly isolated primary MCL cells from patients with relapsed/refractory MCL. In 3H-thymidine incorporation assays, inhibition of CRM1 by KPT185 resulted in a significant dose-dependent growth inhibition of 8 MCL cells, with IC50 values range between 10 nM to 120 nM. The blastoid-variant MCL cell lines (Z-138 and Rec-1) were significantly more sensitive to KPT185 than the non-blastoid variant MCL cell lines. Flow cytometry analysis with fluorescence-labeled Annexin V and propidium iodide showed that KPT185 induced MCL cells apoptosis in both time- and dose-dependent manners, but had no effect on cell cycle arrest. MCL cells treated with KPT185 for 12 hours showed caspase 3 activation and PARP cleavage. As shown in Western blot and confocal microscopy, blocking CRM1 activity by KPT185 in MCL cells up-regulated the protein expression of p53, a known CRM1-mediated export protein, and also induced CRM1 translocation to the nucleus and decreased CRM1 expression. In severe combined immunodeficient (SCID) mice bearing palpable Z-138 tumors, treatment with KPT-276 (similar structure to KPT-185 but improved animal pharmacokinetics), 50mg/kg or 150 mg/kg PO QDx5 each week, or cyclophosphamide 100 mg/kg on days 1–3, was initiated. Tumor growth was significantly inhibited (>75%) in all of treatment groups compared with vehicle control. Neutropenia and other cytotoxic-agent specific effects have not been observed in treated animals. In conclusion, CRM1 inhibitors inhibited growth of MCL cells in vitro and in vivo, and induced apoptosis of MCL cells via inhibition of CRM1 expression and blockage of its translocation with functional nuclear proteins. Our data suggest that novel CRM1 inhibitors provide a potential therapy for patients with relapsed/refractory MCL. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Histone Deacetylase in Waldenström Macroglobulinemia

Blood ◽

10.1182/blood.v112.11.2617.2617 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2617-2617

Author(s):

Xiaoying Jia ◽

Aldo M. Roccaro ◽

Abdel Kareem Azab ◽

Hai T. Ngo ◽

Antonio Sacco ◽

...

Keyword(s):

Bone Marrow ◽

Western Blot ◽

Histone Deacetylase ◽

Cell Lines ◽

Parp Cleavage ◽

Low Grade ◽

Dependent Manner ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence. Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells. Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.

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The PI3K/ERK Dual Inhibitor AEZS-136 Induces ROS-Dependent Necroptotic Cell Death and Exerts Potent Antitumor Effects In NOD/SCID Mice With Hodgkin Lymphoma Cell Line Xenografts

Blood ◽

10.1182/blood.v122.21.3067.3067 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3067-3067

Author(s):

Silvia L Locatelli ◽

Giuliano G Stirparo ◽

Silvia Tartari ◽

Elena Saba ◽

Luca Rubino ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

S Phase ◽

Scid Mice ◽

Ros Generation ◽

Oxygen Species ◽

Dual Inhibitor ◽

Dose Dependent

Abstract Introduction Disease relapse and resistance to chemotherapy represent challenging issues for Hodgkin Lymphoma (HL) patients. PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL patients, thus representing attractive therapeutic targets. Previous results from our phase II study indicate that combining the PI3K/AKT inhibitor perifosine with the RAF/MEK/ERK inhibitor sorafenib can achieve significant clinical responses in relapsed/refractory HL. The present study was therefore aimed at characterizing the in vitro and in vivo activity and mechanism(s) of action of a novel PI3K/ERK dual inhibitor AEZS-136 (Æterna Zentaris GmbH, Germany, EU). Methods Four HL cell lines (L-540, SUP-HD1, KM-H2 and L-428) were used to investigate the in vitro effects of AEZS-136 on cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Live cell imaging experiments were performed to asses the production of reactive oxygen species (ROS). Western blotting (WB) was used to assess the effects of AEZS-136 on MAPK and PI3K/AKT pathways as well as apoptosis. The antitumor efficacy of AEZS-136 was investigated in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results Exposure of L-540, SUP-HD1, KM-H2 and L-428 cell lines to AEZS-136 induced a marked, early and time-dependent dephosphorylation of PI3K/Akt and MAPK pathways that was associated with a significant time and dose-dependent cell growth inhibition [80 ± 3% (mean ±SEM) in the L-540 and SUP-HD1 responsive cell lines] and S phase cell cycle arrest. Indeed, upon AEZS-136 treatment the mean (±SEM) percentages of cells in S phase were reduced by 3-fold (13 ± 1%) as compared to control (33 ± 2%). Significant levels of cell death, as assessed by AnnexinV/PI staining, were only observed in L-540 (62 ± 9 vs 14 ± 3%, P ≤.0001) and SUP-HD1 (46 ± 2% vs 15 ± 2%, P ≤.0001) cell lines and were associated with severe mitochondrial dysfunction (up to 40%, P ≤.001). While no activation of caspase-3 and PARP cleavage were observed in L-540 and SUP-HD1 cells treated with AEZS-136, a potent generation of reactive oxygen species (ROS) was observed upon AEZS-136 treatment (up to 90%, P≤.0001). Pretreating cells with the ROS inhibitor YCG063 strongly inhibited AEZS-136-induced ROS generation, mitochondrial dysfunction and cell death, whereas the pan-caspase inhibitor Z-VADfmk did not. Since ROS generation has been implicated in mediating necroptosis, we tested if blocking programmed necrosis with Necrostatin-1 could prevent AEZS-136-induced cytotoxicity. When L-540 cells were treated with AEZS-136 in the presence of Necrostatin-1, cell death and ROS generation were completely prevented, suggesting that cell death was mechanistically related to necroptosis. Additionally, HL cells responsive to AEZS-136-induced cell death showed a pronounced JNK activation whose inhibition by the JNK inhibitor SP600125 reduced cell death and ROS generation. Furthermore, AEZS-136-increased JNK phosphorylation was inhibited by Necrostatin-1 or YCG063, suggesting that ROS-dependent necroptosis was linked to JNK. Interestingly, GEP analysis of L-540 and SUP-HD1 cell lines, but not KM-H2 and L-428 cells, indicated that AEZS-136 treatment induced upregulation of genes involved in positive regulation of cell death. In addition, in KM-H2 and L-428 cells, AEZS-136 strikingly induced the expression of the immediate early response 3 (IER3). Silencing of IER3 restored sensitivity of KM-H2 and L-428 cells to AEZS-136-induced necroptotic cell death, suggesting that IER3 acts as the signaling molecule that mediated AEZS-136-resistance to oxidative cell death. Finally, in vivo experiments were conducted to investigate the antitumor activity of AEZS-136. Treatment of NOD/SCID mice bearing L540 tumor nodules with increasing dose of AEZS-136 (30 – 60 mg/Kg body weight, PO, 5 days/2 weeks) resulted in a dose-dependent reduction of tumor growth (mean TGI of 70%, P ≤.0001) compared to vehicle-treated controls. No mice experienced any apparent treatment-related toxicity. Conclusions The PI3K/ERK dual inhibitor AEZS-136 demonstrates a potent antitumor activity against HL cell lines by targeting aberrant expression of MAPK and PI3K/Akt pathways. These data support further clinical evaluation of AEZS-136 in refractory/relapsed HL patients. Disclosures: No relevant conflicts of interest to declare.

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Evaluation Of Potency and The Associated Biology Of The Investigational Proteasome Inhibitor, Ixazomib: Redox, Autophagic, and MAPK-Dependent Cell Death In T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cell Lines and Human Lymphoma Xenograft Models

Blood ◽

10.1182/blood.v122.21.4407.4407 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4407-4407 ◽

Cited By ~ 1

Author(s):

Ravi Dashnamoorthy ◽

Irawati Kandela ◽

Savita Bhalla ◽

Andrew Mazar ◽

Andrew M Evens

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Western Blot ◽

Cell Lines ◽

Cathepsin D ◽

Proteasome Inhibitor ◽

Cytotoxic Effect ◽

Xenograft Models ◽

Dose Dependent

Background We investigated the antitumor activity and biologic mechanisms of action of the investigational proteasome inhibitor, ixazomib citrate (MLN2238), in TCL and HL cells with in vitro and in vivo tumor models. Methods TCL cell lines (Jurkat, Hut78 and HH) and HL cell lines (L428, L540, L1236) were treated with ixazomib for 24-48 hours. We analyzed apoptosis and oxidative stress by flow cytometry (FC), and expression of p21, MYC, and MAPK were analyzed with Western blot. For analysis of autophagy, activation of lysosome was examined by fluorescence microscopy using monodansylcadaverine (MDC), which was confirmed by Western blot analysis for Cathepsin D activation and electron microscopy. Additionally, we interrogated signaling pathways with stably transfected shRNA knockouts (KO) +/- ixazomib. In vivo tumor growth inhibition and survival of tumor bearing SCID mice were determined using xenografts derived from Jurkat (TCL) and L540 (HL) cell lines. Results Treatment with 50-100 nanomolar (nM) of ixazomib resulted in time- and dose-dependent cytotoxicity in all cell lines. The IC50 values were 38nM, 52nM, and 41nM for Jurkat, Hut78, and HH, respectively, and 39nM, 60nM, and 117nM for L540, L1236, and L428, respectively. Further, ixazomib resulted in dose-dependent increases in apoptosis by Annexin-V/PI (P<0.001), cleaved PARP, and activation of caspases 3, 8, and 9 in all cell lines. Ixazomib also resulted in increased oxidative stress and decreased intra-cellular glutathione (GSH) and it caused increased p21 expression and degradation of MYC protein. These effects appeared to be redox-dependent as treatment with N-acetyl cysteine (NAC) abrogated apoptosis, oxidative stress, and MYC degradation. Moreover, treatment with ixazomib resulted in cytoplasmic lysosomal abundance with localization in the perinuclear and nuclear region, as observed by MDC staining and electron microscopy in Jurkat and L428. Western blot analysis showed activation of Cathepsin D and presence of Cathepsin D in the nuclear fraction with ixazomib treatment. Furthermore, the autophagic inhibitor, spautin, abrogated cell death induced by ixazomib suggesting a critical role for lysosome directed cytotoxicity with ixazomib-induced cell death. In addition, ixazomib induced several changes in MAPK signaling. In TCL, it increased pERK and p-p38, while pJNK was decreased. Using shRNAs (with ixazomib), ERK and p38 KO had minimal effect in the TCL lines, however JNK KO resulted in increased cell death in Jurkat and Hut78 cells. In HL, ERK and JNK shRNA had minimal effect, while p38 KO increased the cytotoxic effect of ixazomib in all HL cell lines. Finally, in vivo experiments with Jurkat and L540 xenograft-bearing SCID mice showed that ixazomib treatment resulted in significant inhibition of tumor growth (P<0.001) as well as significantly improved survival (P<0.001) versus controls (Figure 1). Conclusions Altogether, ixazomib induced potent cell death at nanomolar and clinically achievable concentrations in TCL and HL cell lines and in vivo human xenograft models. In all cell lines, ixazomib down-regulated MYC and cell death was redox-dependent. Further, autophagy appeared to strongly mediate apoptosis through lysosome directed cytotoxic response. Additionally, in TCL, the cytotoxic effect of ixazomib was mediated through JNK, while p38 was the dominant pathway in HL-related cell death. Continued examination of this novel agent in lymphoma is warranted and rational combinations with MAPK inhibitors should be explored. Disclosures: Evens: Millennium: Consultancy, Honoraria.

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Novel Proteasome Inhibitors Induce Mitochondrial Destabilization and Activate Caspase Mediated Apoptosis In Preclinical Models Of Pediatric B-Cell Cancers

Blood ◽

10.1182/blood.v122.21.5140.5140 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5140-5140

Author(s):

Aneel Paulus ◽

Kasyapa S. Chitta ◽

Eric Sandler ◽

Sharoon Akhtar ◽

Maja Kuranz ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Cell Lines ◽

Response Rate ◽

Proteasome Inhibitors ◽

Preclinical Models ◽

Raji Cells ◽

Dose Dependent ◽

Mts Assay

Abstract Background In the U.S. over 14,000 new cancer cases occur annually in people under 21 years of age; approximately 21% of cases are acute lymphoblastic leukemia (ALL) and ∼ 30% classify as Burkitt lymphoma (BL). Most cases of ALL and BL are due to malignant B-cells that have acquired disease-defining oncogenic lesions that facilitate their rapid proliferation. To maintain a state of homeostasis, the malignant cells rely heavily on the ability of the internal degradation and recycling systems to match the high rate of protein turnover. As such, the ubiquitin proteasome system (UPS) is central in this operation where it prevents buildup of misfolded proteins while also regulating the availability of anti-apoptotic proteins such as BCL2 and MCL1. These observations coupled with the observation that UPS disruption leads to cellular demise have resulted in the development of several proteasome inhibitors (PI). These PI have primarily been examined in adult B-cell malignancies. The feasibility of a bortezomib-containing regimen for use in pediatric malignancies has been examined by the TACL consortium and demonstrated a 73% response rate in relapsed/refractory ALL patients. However, a major limitation of bortezomib-based therapy is an increased incidence of peripheral neuropathy (PN), which was also noted in the TACL study. As such, more potent and reportedly less toxic PI have been developed that seem to carry considerably lower risk for development of PN. Such agents that maintain a high response rate and carry reduced long-term adverse effects are highly desirable in pediatric age patients. Aim Investigation of the novel proteasome inhibitors carfilzomib and MLN9708 (Millennium Pharmaceuticals) in preclinical models of pediatric B-lymphocytic cancer. Methods Representative preclinical models of BL (Raji cell line) and B-ALL (ALL-1 cells) were used in this study. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Cell viability was measured by MTS assay. Apoptosis was determined by annexin-v/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was performed via immunoblot. Results In an effort to understand the effect of carfilzomib and MLN9708 in our pediatric cancer models, we first assessed their ability to inhibit proteasomal activity. While chymotrypsin-like activity, which is the target of both PIs, is inhibited by more than 90% in both cell lines, inhibition (∼ 35%) of caspase-like function conferred by the b1 subunit was more significantly pronounced in MLN9708 treated Raji and ALL-1 cells. Next we performed an MTS assay to determine the growth inhibitory effects of these PI in vitro. Both PI inhibited the growth of the Raji and ALL-1 cells in a dose dependent manner. Carfilzomib showed a more potent effect (IC50 ∼1nM in both cell lines) as compared to MLN9708 (IC50 ∼30nM in ALL-1 cells and ∼50nM in Raji cells). To further understand mechanisms of growth inhibition we examined both tumor cell lines for induction of apoptosis along with corresponding markers. As compared to Raji cells, we observed ALL-1 cells to be more sensitive to the effects of both PI, albeit with MLN9708 (50nM) inducing more cell death (65%) as compared to carfilzomib (10nM, 50% cell death). Further, we observed altered MOMP, cleavage of caspases 9, 3 and PARP-1 were found to occur in both cell lines in presence of carfilzomib or MLN9708. These changes indicate the apoptotic effects of novel PI in ALL-1 and Raji cells are via mitochondrial destabilization and caspase activation. As inhibition of the UPS aids NOXA and BIM (pro-apoptotic BCL2 proteins) mediated cell death, we examined the expression of BCL2 family proteins changes in Raji and ALL-1 cells in response to novel PI. Indeed protein levels of both BCL2 and MCL1 were reduced in carfilzomib and MLN9708 treated cells. Reduced BCL2 expression was more pronounced in ALL-1 cells as compared to Raji cells and downregulation was noted to be dose-dependent. Summary Our data show that both carfilzomib and MLN9708 potently inhibit the viability of malignant ALL and BL cells in vitro. We conclude that PI are promising anti-neoplastic therapeutics that target not only the UPS but also modulate the expression of critical anti-apoptotic proteins. Data from mechanistic studies conducted herein underscore the need for further research in in vivo models of these diseases. Disclosures: Foran: Celgene: Research Funding.

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Effect of sunitinib and pazopanib on necrosis and autophagic cell death in cancer cells: Role of cathepsin B.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e15513 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e15513-e15513 ◽

Cited By ~ 1

Author(s):

Matteo Santoni ◽

Consuelo Amantini ◽

Maria Beatrice Morelli ◽

Valerio Farfariello ◽

Massimo Nabissi ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Cancer Cells ◽

Cell Lines ◽

Cathepsin B ◽

Kinase Inhibitors ◽

Autophagic Cell Death ◽

Dependent Manner ◽

Acridine Orange Staining

e15513 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on TKI-induced cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the anti-proliferative and cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrotic cell death was evaluated by Annexin-V/PI staining and FACS analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sunitinib and pazopanib markedly reduced at mM dose the viability of BC cells. Treatment for 24h with 20µM of sunitinib, by triggering “Incomplete autophagy”, induced necrosis of BC cells. In addition, sunitinib as a lysosomotropic agent, entered free within the lysosomes, where by increasing lysosomal pH and impairing cathepsin B activity, induced lysosomal-dependent necrosis. By contrast, treatment of BC cells for 72h with 20µM of pazopanib induced autophagic cell death, which was markedly reversed in a dose-dependent manner by the autophagic inhibitor 3-MA. The pazopanib-induced autophagic cell death was associated with increased procathepsin B cleavage and enhanced cathepsin B activity. Conclusions: Overall, our results show different cathepsin B-dependent cancer cell death mechanisms induced by sunitinib or pazopanib, providing the biological basis for novel molecularly targeted approaches.

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Resveratrol Exerts Antiproliferative Effect and Induces Apoptosis in Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v110.11.1383.1383 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1383-1383

Author(s):

Aldo Roccaro ◽

Xavier Leleu ◽

Anne-Sophie Moreau ◽

Antonio Sacco ◽

Lian Xu ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Flow Cytometry ◽

Western Blot ◽

Cell Lines ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Downstream Target ◽

Dose Dependent

Abstract Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom’s Macroglobulinemia (WM). Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol. Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib. Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

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The synthetic opioid fentanyl enhances viral replication in vitro

PLoS ONE ◽

10.1371/journal.pone.0249581 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249581

Author(s):

Ling Kong ◽

Rebekah Karns ◽

Mohamed Tarek M. Shata ◽

Jennifer L. Brown ◽

Michael S. Lyons ◽

...

Keyword(s):

Cell Death ◽

Viral Replication ◽

Cell Lines ◽

Replicative Fitness ◽

Synthetic Opioids ◽

The Impact ◽

Dose Dependent Increase ◽

Dose Dependent ◽

Higher Doses

The US is in the midst of a major drug epidemic fueled in large part by the widespread recreational use of synthetic opioids such as fentanyl. Persons with opioid use disorder are at significant risk for transmission of injection-associated infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV). Commonly abused substances may antagonize immune responses and promote viral replication. However, the impact of synthetic opioids on virus replication has not been well explored. Thus, we evaluated the impact of fentanyl and carfentanil using in vitro systems that replicate infectious viruses. Fentanyl was used in cell lines replicating HBV or HCV at concentrations of 1 ng, 100 ng, and 10 ug. Viral protein synthesis was quantified by ELISA, while apoptosis and cell death were measured by M30 or MTT assays, respectively. HCV replicative fitness was evaluated in a luciferase-based system. RNAseq was performed to evaluate cellular gene regulation in the presence of fentanyl. Low dose fentanyl had no impact on HCV replication in Huh7.5JFH1 hepatocytes; however, higher doses significantly enhanced HCV replication. Similarly, a dose-dependent increase in HCV replicative fitness was observed in the presence of fentanyl. In the HepG2.2.15 hepatocyte cell line, fentanyl caused a dose-dependent increase in HBV replication, although only a higher doses than for HCV. Addition of fentanyl resulted in significant apoptosis in both hepatocyte cell lines. Cell death was minimal at low drug concentrations. RNAseq identified a number of hepatocyte genes that were differentially regulated by fentanyl, including those related to apoptosis, the antiviral / interferon response, chemokine signaling, and NFκB signaling. Collectively, these data suggest that synthetic opioids promote viral replication but may have distinct effects depending on the drug dose and the viral target. As higher viral loads are associated with pathogenesis and virus transmission, additional research is essential to an enhanced understanding of opioid-virus pathogenesis and for the development of new and optimized treatment strategies.

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