Investigation of Lupeol as Anti-Melanoma Agent: An In Vitro-In Ovo Perspective

Malignant melanoma (MM) represents the most life-threatening skin cancer worldwide, with a narrow and inefficient chemotherapeutic arsenal available in advanced disease stages. Lupeol (LUP) is a triterpenoid-type phytochemical possessing a broad spectrum of pharmacological properties, including a potent anticancer effect against several neoplasms (e.g., colorectal, lung, and liver). However, its potential as an anti-melanoma agent has been investigated to a lesser extent. The current study focused on exploring the impact of LUP against two human MM cell lines (A375 and RPMI-7951) in terms of cell viability, confluence, morphology, cytoskeletal distribution, nuclear aspect, and migration. Additionally, the in ovo antiangiogenic effect has been also examined. The in vitro results indicated concentration-dependent and selective cytotoxicity against both MM cell lines, with estimated IC50 values of 66.59 ± 2.20 for A375, and 45.54 ± 1.48 for RPMI-7951, respectively, accompanied by a reduced cell confluence, apoptosis-specific nuclear features, reorganization of cytoskeletal components, and inhibited cell migration. In ovo, LUP interfered with the process of angiogenesis by reducing the formation of neovascularization. Despite the potential anti-melanoma effect illustrated in our in vitro-in ovo study, further investigations are required to elucidate the underlying LUP-induced effects in A375 and RPMI-7951 MM cells.

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The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Blood ◽

10.1182/blood.v126.23.5350.5350 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5350-5350

Author(s):

Mairead Reidy ◽

Marianne VanDijk ◽

Michael O'Neill ◽

Michael O'Dwyer

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Surface Expression ◽

Down Regulation ◽

Pim Kinases ◽

Ic50 Values ◽

And Migration

Abstract Background: The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration. Methods: We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining. To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5. Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C. Results: Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1). At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged. Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc. IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2). To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient. Conclusion: Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures O'Neill: Inflection Biosciences: Employment.

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Synthesis and Antitumor Activity Evaluation of New Phenanthrene-Based Tylophorine Derivatives

Letters in Organic Chemistry ◽

10.2174/1570178615666181025115513 ◽

2019 ◽

Vol 16 (6) ◽

pp. 462-467

Author(s):

Songtao Li ◽

Hongling Zhao ◽

Zhifeng Yin ◽

Shuhua Deng ◽

Yang Gao ◽

...

Keyword(s):

Antitumor Activity ◽

Cell Lines ◽

Antitumor Activities ◽

Human Carcinoma ◽

Carcinoma Cell Lines ◽

Structure Activity ◽

Human Carcinoma Cell ◽

Ic50 Values ◽

Relationship Of

A series of new phenanthrene-based tylophorine derivatives (PBTs) were synthesized in good yield and their structures were characterized by 1H-NMR spectroscopy and ESI MS. In vitro antitumor activity of these compounds against five human carcinoma cell lines, including HCT116 (colorectal), BGC-823 (gastric), HepG-2 (hepatic), Hela (cervical) and H460 (lung) cells, was evaluated by MTT assay. Among these PBTs, compound 6b showed the highest antitumor activities against HCT116 and HepG-2 cell lines with IC50 values of 6.1 and 6.4 μM, respectively, which were comparable to that of adriamycin hydrochloride. The structure-activity relationship of these compounds was also discussed based on the results of their antitumor activity.

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Delivery of melittin-loaded niosomes for breast cancer treatment: an in vitro and in vivo evaluation of anti-cancer effect

Cancer Nanotechnology ◽

10.1186/s12645-021-00085-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Farnaz Dabbagh Moghaddam ◽

Iman Akbarzadeh ◽

Ehsan Marzbankia ◽

Mahsa Farid ◽

Leila khaledi ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Treatment ◽

Cell Lines ◽

Encapsulation Efficiency ◽

Inbred Mice ◽

Breast Cancer Treatment ◽

Anticancer Effect ◽

Anti Cancer

Abstract Background Melittin, a peptide component of honey bee venom, is an appealing candidate for cancer therapy. In the current study, melittin, melittin-loaded niosome, and empty niosome had been optimized and the anticancer effect assessed in vitro on 4T1 and SKBR3 breast cell lines and in vivo on BALB/C inbred mice. "Thin-layer hydration method" was used for preparing the niosomes; different niosomal formulations of melittin were prepared and characterized in terms of morphology, size, polydispersity index, encapsulation efficiency, release kinetics, and stability. A niosome was formulated and loaded with melittin as a promising drug carrier system for chemotherapy of the breast cancer cells. Hemolysis, apoptosis, cell cytotoxicity, invasion and migration of selected concentrations of melittin, and melittin-loaded niosome were evaluated on 4T1 and SKBR3 cells using hemolytic activity assay, flow cytometry, MTT assay, soft agar colony assay, and wound healing assay. Real-time PCR was used to determine the gene expression. 40 BALB/c inbred mice were used; then, the histopathology, P53 immunohistochemical assay and estimate of renal and liver enzyme activity for all groups had been done. Results This study showed melittin-loaded niosome is an excellent substitute in breast cancer treatment due to enhanced targeting, encapsulation efficiency, PDI, and release rate and shows a high anticancer effect on cell lines. The melittin-loaded niosome affects the genes expression by studied cells were higher than other samples; down-regulates the expression of Bcl2, MMP2, and MMP9 genes while they up-regulate the expression of Bax, Caspase3 and Caspase9 genes. They have also enhanced the apoptosis rate and inhibited cell migration, invasion in both cell lines compared to the melittin samples. Results of histopathology showed reduce mitosis index, invasion and pleomorphism in melittin-loaded niosome. Renal and hepatic biomarker activity did not significantly differ in melittin-loaded niosome and melittin compared to healthy control. In immunohistochemistry, P53 expression did not show a significant change in all groups. Conclusions Our study successfully declares that melittin-loaded niosome had more anti-cancer effects than free melittin. This project has demonstrated that niosomes are suitable vesicle carriers for melittin, compare to the free form.

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YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

International Journal of Molecular Sciences ◽

10.3390/ijms22137226 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7226

Author(s):

Violeta Stojanovska ◽

Aneri Shah ◽

Katja Woidacki ◽

Florence Fischer ◽

Mario Bauer ◽

...

Keyword(s):

Cell Lines ◽

Cancer Progression ◽

Cell Function ◽

Trophoblast Cell ◽

Mrna Levels ◽

Trophoblast Cells ◽

Invasion And Migration ◽

And Migration ◽

Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

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Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽

10.3233/tub-200072 ◽

2021 ◽

Vol 43 (1) ◽

pp. 11-26

Author(s):

Maike Busch ◽

Natalia Miroschnikov ◽

Jaroslaw Thomas Dankert ◽

Marc Wiesehöfer ◽

Klaus Metz ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Cancer Progression ◽

Treated Patient ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Gene Assay ◽

The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽

10.3390/cells10081870 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1870

Author(s):

Klaudia Skrzypek ◽

Grażyna Adamek ◽

Marta Kot ◽

Bogna Badyra ◽

Marcin Majka

Keyword(s):

Transcription Factor ◽

Cell Lines ◽

Migration Activity ◽

Id Proteins ◽

Rh30 Cell ◽

Str Profile ◽

And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

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In Vitro Pharmaco-Toxicological Characterization of Melissa officinalis Total Extract Using Oral, Pharynx and Colorectal Carcinoma Cell Lines

Processes ◽

10.3390/pr9050850 ◽

2021 ◽

Vol 9 (5) ◽

pp. 850

Author(s):

Kristine Guran ◽

Roxana Buzatu ◽

Iulia Pinzaru ◽

Madalina Boruga ◽

Iasmina Marcovici ◽

...

Keyword(s):

Colorectal Carcinoma ◽

Melissa Officinalis ◽

Pharmacological Profile ◽

In Ovo ◽

Total Extract ◽

Antiangiogenic Effect ◽

Beneficial Effects ◽

Gram Positive Bacteria ◽

Potential Benefits

Melissa officinalis is a medicinal herb with an extensive pharmacological profile that has been proven to have beneficial effects in oral and gastrointestinal disorders. However, the effects of this plant in oral, pharyngeal, and colorectal malignancies, types of cancer with an increased incidence in recent years, are less investigated. The present study aims to evaluate the pharmacological profile of a Melissa officinalis total extract for potential benefits in oral, pharynx and colorectal carcinoma. The LC-MS profile of MO total extract (MOte) indicated a rich content in polyphenols, data that support the potent antioxidant capacity exhibited and the antimicrobial activity against both Gram-negative and Gram-positive bacteria. In addition, MOte triggered a dose-dependent and selective decrease in the viability of tumor cells (tongue and pharynx squamous cell carcinomas, and colorectal adenocarcinoma), with the most significant effect being recorded at 100 µg/mL. At the same concentration, MOte exhibited an antiangiogenic effect by inhibiting the process of angiogenesis in ovo. Overall, our findings support the potential benefits of Melissa officinalis leaf total extract as a valuable candidate for the prophylaxis of oral, pharyngeal and colorectal neoplasms.

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EXTH-64. SMALL GTPASES IN MENINGIOMAS: PROLIFERATION, MIGRATION, SURVIVAL, POTENTIAL TREATMENT AND INTERACTIONS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.418 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii101-ii101

Author(s):

Christoph Kesseler ◽

Julian Kahr ◽

Natalie Waldt ◽

Nele Stroscher ◽

Josephine Liebig ◽

...

Keyword(s):

Overall Survival ◽

Cell Lines ◽

Small Gtpases ◽

Potential Treatment ◽

Rock Inhibitor ◽

Meningioma Cell ◽

And Migration ◽

Proliferation And Migration

Abstract PURPOSE To evaluate the role of the small GTPases RhoA, Rac1 and Cdc42 in meningiomas as therapeutic targets and their interactions in meningiomas. EXPERIMENTAL DESIGN We analyzed expression of GTPases in human meningioma samples and meningioma cell lines of various WHO grades. Malignant IOMM-Lee meningioma cells were used to generate shRNA mediated knockdowns of GTPases RhoA, Rac1 or Cdc42 and to study knockdown effects on proliferation and migration, as well as analysis of cell morphology by confocal microscopy. The same tests were used to investigate effects of the two inhibitors Fasudil and EHT-1864 of malignant IOMM-Lee, KT21 and benign Ben-Men cells and the effects of these drugs on IOMM-Lee knockdown cells. The effects of GTPase knockdowns and Fasudil treatment were studied in terms of overall survival by intracranial xenografts of mice. Potential interactions of GTPases regarding NF2, mTOR and FAK-Paxillin were examined. RESULTS Small GTPases were upregulated in meningiomas of higher tumor grades. Reduced proliferation and migration could be achieved by GTPase knockdown in IOMM-Lee cells. Additionally, the ROCK-inhibitor Fasudil and Rac1-inhibitor EHT-1864 reduced proliferation in different meningioma cell lines and reduced proliferation and migration independent of GTPase knockdowns/status. Moreover, overall survival in vivo could also be increased by knockdowns of RhoA and Rac1 as well as Fasudil treatment. GTPase expression was affected dependent on the NF2 status but effects were not very distinct, indicating that NF2 is not strongly involved in GTPase regulation in meningiomas. In terms of mTOR and FAK-Paxillin signaling, each GTPase changes those pathways in a different manner. CONCLUSION Small GTPases are important effectors in meningioma proliferation and migration in vitro as well as survival in vivo and their inhibition should be considered as potential treatment option.

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BSCI-12. Inhibition of melanoma brain metastasis by targeting miR-146a

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab071.011 ◽

2021 ◽

Vol 3 (Supplement_3) ◽

pp. iii3-iii3

Author(s):

Jiwei Wang ◽

Emma Rigg ◽

Taral R Lunavat ◽

Wenjing Zhou ◽

Zichao Feng ◽

...

Keyword(s):

Brain Metastasis ◽

Tumor Cells ◽

Cell Lines ◽

Western Blots ◽

Cell Growth And Migration ◽

Human Astrocytes ◽

And Migration ◽

The Brain

Abstract Background Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released by the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. Materials and Methods miRNAs from exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. Results miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146a in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. Conclusions MiR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

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Six New neo-Clerodane Diterpenoids from Aerial Parts of Scutellaria barbata and Their Cytotoxic Activities

Planta Medica ◽

10.1055/a-0638-8255 ◽

2018 ◽

Vol 84 (17) ◽

pp. 1292-1299 ◽

Cited By ~ 9

Author(s):

Guo-Chun Yang ◽

Jia-Hui Hu ◽

Bing-Long Li ◽

Huan Liu ◽

Jia-Yue Wang ◽

...

Keyword(s):

Cell Lines ◽

Human Cancer ◽

In Vitro Cytotoxicity ◽

Cytotoxic Activities ◽

Scutellaria Barbata ◽

Human Cancer Cell Lines ◽

Aerial Parts ◽

Ic50 Values ◽

Clerodane Diterpenoids

AbstractSix new neo-clerodane diterpenoids (1–6), scutebatas X – Z, A1-C1, along with twelve known ones (7–18) were obtained via the phytochemical investigation of the aerial parts of Scutellaria barbata. Their structures were established by detailed spectroscopic analysis. The absolute configurations of 1 and 2, as the representative members of this type, were identified based on a circular dichroic exciton chirality method. Moreover, in vitro cytotoxicity of compounds 1–6 were evaluated against three human cancer cell lines (SGC-7901, MCF-7, and A-549) using the MTT method. Compound 6 showed cytotoxic activities against all the three cell lines with IC50 values of 17.9, 29.9, and 35.7 µM, respectively.

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