Human Prostate Carcinoma Cell Line Research Articles

Abstract 2824: Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line

Abstract Prostate cancer is the second most common cancer in men with new cases diagnosed in 1.4 million men each year. Luckily the 5-year survival rates in developed countries are high reaching over 90%. However, approximately one third will develop a metastatic disease. In metastatic patients, prostate cancer almost always progresses despite the androgen deprivation therapy and is then referred to as “castration-resistant (mCRPC)”. Bone is involved in 80% of patients with metastasis, and the bone lesions are typically osteoblastic. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). They also express low levels of prostate-specific membrane antigen (PSMA). The aim of this study was to establish orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells (ATCC) were inoculated orthotopically into the prostate of NOG mice, and 5 × 105 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. Tumor growth was followed by PSA measurements every second week using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. The observed tumor take rate was 92% in the orthotopic and 100% in the intratibial models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. Taken together, the 22Rv1 prostate cancer models are relatively fast growing, express AR and present good take rates. Furthermore, the intratibial model represents robust tumor-induced changes in bone with strong osteoblastic component. Thus, these models have several advantages in efficacy studies of novel drug candidates for prostate cancer. Citation Format: Justyna Zdrojewska, Katja Fagerlund, Jenni H. Mäki-Jouppila, Mari I. Suominen. Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2824.

Abstract 2858: Radiotherapy and NK cell-based immunotherapy: An evolutionary double-bind

Abstract Evolution-informed therapies exploit the ecological and evolutionary consequences of drug resistance to inhibit the expansion of treatment resistant populations and prolong time to progression. One strategy, termed an evolutionary double-bind, uses an initial therapy to elicit a specific adaptive mechanism that is then selectively targeted by a follow-on therapy. Here we examine combining radiation therapy followed by immunotherapy as a double bind strategy. Radiotherapy (RT) induces lethal double-strand DNA breaks in tumor cells and radiosensitivity is governed by the cells’ DNA repair capabilities. Radioresistant cancer cells upregulate DNA damage response pathways. However, successful adaptations to RT and other DNA damaging agents can increase natural killer (NK) cell ligand expression on tumor cells increasing their vulnerability to the immune system. We conducted a multidisciplinary investigation of this potential evolutionary double bind using an isogenic cell line model of radiation resistance derived from an androgen-independent human prostate carcinoma cell line (22Rv1). We demonstrate that RT-resistant cells maintain higher expression of NK cell ligands and have a 2-fold increase in sensitivity to NK cell mediated killing. Furthermore, we show that preferential targeting of the RT-resistant population by NK cells is preserved in competition assays with varying initial population frequencies. Scope of treatment responses are dependent on population frequencies in our in vitro analysis. Mathematical modeling quantified three key elements of a double-bind: cost of resistance, inter-specific competition between RT-sensitive and RT-resistant lines, and preferential targeting of RT-resistant lines by NK cells. We conclude that RT followed by immunotherapy produces an evolutionary double bind that can be exploited in heterogenous tumors to limit RT resistance. Furthermore, model simulations predict that extinction of both populations is achievable by sequential RT and NK cell therapy. Citation Format: Kimberly Luddy, Jeffery West, Mark Robertson-Tessi, Bina Desai, Taylor M. Bursell, Sarah Barrett, Joel Brown, Jacintha O'Sullivan, Robert Gatenby, Laure Marignol, Alexander R. Anderson, Cliona O'Farrelly. Radiotherapy and NK cell-based immunotherapy: An evolutionary double-bind [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2858.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Structures of Mammeasins P and Q, Coumarin-Related Polysubstituted Benzofurans, from the Thai Medicinal Plant Mammea siamensis (Miq.) T. Anders.: Anti-Proliferative Activity of Coumarin Constituents against Human Prostate Carcinoma Cell Line LNCaP.

A methanol extract of the flowers of Mammea siamensis (Miq.) T. Anders. (Calophyllaceae) showed anti-proliferative activity against human prostate carcinoma LNCaP cells (IC50 = 2.0 µg/mL). Two new coumarin-related polysubstituted benzofurans, mammeasins P (1) and Q (2), and a known polysubstituted coumarin mammea B/AC cyclo F (39) were isolated from the extract along with 44 previously reported polysubstituted coumarin constituents (3-38 and 40-47). The structures of two new compounds (1 and 2) were determined based on their spectroscopic properties derived from the physicochemical evidence including NMR and MS analyses and taking the plausible generative pathway into account. Among the coumarin constituents, mammeasins A (3, IC50 = 1.2 µM) and B (4, 0.63 µM), sugangin B (18, 1.5 µM), kayeassamins E (24, 3.0 µM) and G (26, 3.5 µM), and mammeas E/BA (40, 0.88 µM), E/BB (41, 0.52 µM), and E/BC (42, 0.12 µM) showed relatively potent anti-proliferative activity.

Functional analysis of human olfactory receptors with a high basal activity using LNCaP cell line.

Humans use a family of more than 400 olfactory receptors (ORs) to detect odorants. However, deorphanization of ORs is a critical issue because the functional properties of more than 80% of ORs remain unknown, thus, hampering our understanding of the relationship between receptor function and perception. HEK293 cells are the most commonly used heterologous expression system to determine the function of a given OR; however, they cannot functionally express a majority of ORs probably due to a lack of factor(s) required in cells in which ORs function endogenously. Interestingly, ORs have been known to be expressed in a variety of cells outside the nose and play critical physiological roles. These findings prompted us to test the capacity of cells to functionally express a specific repertoire of ORs. In this study, we selected three cell lines that endogenously express functional ORs. We demonstrated that human prostate carcinoma (LNCaP) cell lines successfully identified novel ligands for ORs that were not recognized when expressed in HEK293 cells. Further experiments suggested that the LNCaP cell line was effective for functional expression of ORs, especially with a high basal activity, which impeded the sensitive detection of ligand-mediated activity of ORs. This report provides an efficient functional assay system for a specific repertoire of ORs that cannot be characterized in current cell systems.

In Vitro Assessment of Antistaphylococci, Antitumor, Immunological and Structural Characterization of Acidic Bioactive Exopolysaccharides from Marine Bacillus cereus Isolated from Saudi Arabia

A strain of Bacillus cereus was isolated from the Saudi Red Sea coast and identified based on culture features, biochemical characteristics, and phylogenetic analysis of 16S rRNA sequences. EPSR3 was a major fraction of exopolysaccharides (EPS) containing no sulfate and had uronic acid (28.7%). The monosaccharide composition of these fractions is composed of glucose, galacturonic acid, and arabinose with a molar ratio of 2.0: 0.8: 1.0, respectively. EPSR3 was subjected to antioxidant, antitumor, and anti-inflammatory activities. The results revealed that the whole antioxidant activity was 90.4 ± 1.6% at 1500 µg/mL after 120 min. So, the IC50 value against DPPH radical found about 500 µg/mL after 60 min. While using H2O2, the scavenging activity was 75.1 ± 1.9% at 1500 µg/mL after 60 min. The IC50 value against H2O2 radical found about 1500 µg/mL after 15 min. EPSR3 anticytotoxic effect on the proliferation of (Bladder carcinoma cell line) (T-24), (human breast carcinoma cell line) (MCF-7), and (human prostate carcinoma cell line) (PC-3) cells. The calculated IC50 for cell line T-24 was 121 ± 4.1 µg/mL, while the IC50 for cell line MCF-7 was 55.7 ± 2.3 µg/mL, and PC-3 was 61.4 ± 2.6 µg/mL. Anti-inflammatory activity was determined for EPSR3 using different methods as Lipoxygenase (LOX) inhibitory assay gave IC50 12.9 ± 1.3 µg/mL. While cyclooxygenase (COX-2) inhibitory test showed 29.6 ± 0.89 µg /mL. EPSR3 showed potent inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci. The exposure times of EPSR3 for the complete inhibition of cell viability of methicillin resistant S. aureus was found to be 5% at 60 min. Membrane stabilization inhibitory gave 35.4 ± 0.67 µg/mL. EPSR3 has antitumor activity with a reasonable margin of safety. The antitumor activity of EPSR3 may be attributed to its content from uronic acids with potential for cellular antioxidant and anticancer functional properties.

Investigation of the cytotoxicity induced by didocosahexaenoin, an omega 3 derivative, in human prostate carcinoma cell lines

The aim of the present study was to investigate the cytotoxicity induced by an omega-3 derivative, didocosahexaenoin (Dido) on human prostate carcinoma cells and to compare the cytotoxicity to that of docosahexaenoic acid (DHA). Different carcinoma- and non-carcinoma cells were exposed to various concentrations of omega-3 compounds at varying exposure times and the cytotoxicity was measured by MTT assay. The mechanism of Dido-induced apoptosis was investigated in prostate carcinoma cells. Dido induced stronger cytotoxicity than DHA in human prostate carcinoma cells in a dose- and time-dependent manner. Dido was also more selective and potent in inducing cytotoxicity in prostate carcinoma cells than other carcinoma cell lines tested. Pre-treatment with Dido increased the level of reactive oxygen species (ROS) in prostate carcinoma cells. Pre-treatment with various antioxidants reduced the cytotoxicity induced by Dido. Pre-treatment with Dido ≥30 ​μM also induced apoptosis which was suggested to involve an externalisation of phosphatidyl serine, a significant increase in the mitochondrial membrane potential (p ​< ​0.01) and the level of activated caspase 3/7 (p ​< ​0.05) in prostate carcinoma cells. This study is the first to show that Dido induced cytotoxicity with high selectivity and higher potency than DHA in human prostate carcinoma cells. The mechanism of action is likely to involve an increase in the level of ROS, loss in the mitochondrial membrane potential as well as externalisation of phosphatidyl serine and increase in the caspase 3/7 activity. Dido may have potential to be used for the adjuvant therapy or combination therapy with conventional chemotherapeutic drugs.

Therapeutic Multidose Preparation of a Ready-to-Use 177Lu-PSMA-617 Using Carrier Added Lutetium-177 in a Hospital Radiopharmacy and Its Clinical Efficacy.

Introduction: [177Lu]Lu-prostate-specific membrane antigen (PSMA)-617 has emerged as a promising radiopharmaceutical for targeting PSMA in metastatic castrate-resistant prostate carcinoma (mCRPC). We have optimized the radiolabeling protocol for a multidose formulation (27-28.8 GBq equivalent to 6-7 patient-doses) of [177Lu]Lu-PSMA-617 using [177Lu]Lu3+ produced via 176Lu(n,γ)177Lu route with moderate specific activity (0.66-0.81 GBq/μg). Methods: [177Lu]Lu-PSMA-617 was synthesized using moderate specific activity [177Lu]LuCl3 (0.74 GBq/μg) with PSMA-617 having metal-to-ligand molar ratio ∼1: 2.5 in CH3COONH4 buffer (0.1 M) containing gentisic acid at pH 4.0-4.5. Human prostate carcinoma cell line LNCaP cell (high PSMA expression) was used for in vitro cell-binding studies and generating tumor xenograft models in nude mice for tissue biodistribution studies. Several batches of the present formulation have been clinically administered in mCRPC patients (single patient dose: 4.44-5.55 GBq per cycle). Results: In this study we report a consistent and reproducible protocol for multidose formulations of [177Lu]Lu-PSMA-617 for adopting in a hospital radiopharmacy setting. Although the radiochemical yield of [177Lu]Lu-PSMA-617 was found to be 97.30% ± 1.03%, the radiochemical purity was 98.24% ± 0.50% (n = 19). In vitro and serum stability of [177Lu]Lu-PSMA-617 was retained up to 72 and 120 h after radiolabeling and upon storage at -20°C with a radioactive concentration between 0.37 and 0.74 GBq/mL upon using stabilizer concentration as low as 43-48 μg/mCi. Preclinical cell-binding studies of [177Lu]Lu-PSMA-617 revealed specific binding with LNCaP cells of 17.4% ± 2.4%. The uptake in LnCaP xenografted tumor (nude mice) was 7.5 ± 2.6% ID/g for ∼1.5-2.0 cm3 tumor volume at 24-h post-injection. Post-therapy (24 h) SPECT image of mCRPC patients with prior orchidectomy and various hormone therapy showed specific localization of [177Lu]Lu-PSMA-617 in the tumor region. Conclusions: Formulation of a ready-to-use multidose formulation of [177Lu]Lu-PSMA-617 was successfully achieved and the procedure was optimized for routine preparation at a hospital radiopharmacy set-up. High degree of localization of [177Lu]Lu-PSMA-617 in post-therapy SPECT scan and the post-therapeutic response confirms its therapeutic efficacy. Clinical Trials.gov ID: RPC/51/Minutes/Final dated 16th October, 2019.

The Synthesis of Conjugated Peptides Containing Triazole and Quinolone-3-Carboxamide Moieties Designed as Anticancer Agents.

Background: Cancer is a major health concern in human populations worldwide, and due to its causes being multi-factorial, it is not easily curable. Many attempts have been made to tackle this disease in hopes of finding effective anticancer agents which are not harmful to healthy tissues. Peptides with several medicinal activities have been shown to be good candidates as anticancer agents to replace common classic anticancer drugs. Peptides in conjugation with either biologically active heterocyclic compounds or anticancer drugs may result in new molecules compiling the biological benefits of both individual compounds within a unit structure.Objective: In this study some triazole-peptide conjugates as well as ciprofloxacin-peptide conjugates were designed, synthesized, and their anticancer activities evaluated. A normal skin cell line, NIH3, was also employed to determine the safety profiles of these conjugates.Materials and Methods: Two peptides; YIGSR and LSGNK were synthesized by the solid phase peptide synthesis (SPPS) method using Wang resin. Cell viability was examined by employing the MTT assay. To determine the cytotoxicity of the triazole and ciprofloxacin conjugates, two human cancer cell lines were employed; HepG2 (human liver cancer cell line) and LNCaP (human prostatic carcinoma cell line). A human skin fibroblast cell line was also included for comparison.Results: MTT results showed that all the compounds could inhibit the viability of cancerous cells in a concentration- dependent manner.Conclusions: The results showed that these peptide conjugates are toxic against the aforementioned cancerous cells and thus may raise a hope for finding new anticancer agents made by such strategy in the near future.

Anticancer, anticholinesterase and antidiabetic activities of tunceli garlic (Allium tuncelianum): determining its phytochemical content by LC–MS/MS analysis

We aimed to investigate the phytochemical content and pharmaceutical activity of methanol extracts of Allium tuncelianum (MAT). In this study, phytochemical contents of MAT were analyzed by liquid chromatography mass-spectrometry (LC–MS/MS) technique. Anticancer activity was determined on six different cell lines. By flow cytometric method, apoptotic activity was examined with annexin V in human prostate carcinoma (PC3) cell lines where the highest cytotoxic activity was observed. Real Time PCR was used to investigate the molecular genetic size of the effect. Anticholinesterase and antidiabetic activities were performed using enzyme inhibition technique. Enzyme inhibitory activity of MAT was determined using AChE, α-glucosidase enzymes. In these extracts, “μg Analyte/g MAT” as its unit; quinic acid (6458.45 ± 2.389), malic acid (4187.25 ± 0.544) and vanilic acid (349.4 ± 0.052) were found to be major components. The highest cytotoxic activity was found in PC3 cells (IC50: 105.72 µg/mL). Therefore, the mechanism of apoptotic effect in this cell line was also investigated. In a statistically meaningful proportion, MAT increased Caspase 3 and Caspase 9 gene expressions and suppressed IL6, IL8, MMP2 and MMP9 gene expressions. On the other hand, MAT demonstrated effective inhibition effects against AChE (IC50: 11.25 μg/mL), and α-glycosidase (IC50: 9.85 μg/mL) enzymes. As a result, it can be argued that Allium tuncelianum can be used as an alternative treatment for prostate cancer, diabetes and some neurodegenerative diseases.

Salvage chemotherapy with cisplatin, ifosfamide, and paclitaxel in metastatic castration-resistant prostate cancer.

123 Background: Recent developments in the treatment of metastatic castration resistant prostate cancer (mCRPC) lead to a revival of platinum-based chemotherapy demonstrating increased activity in patients with aggressive variants of disease. Here, we report on the results of a combinational salvage therapy with cisplatin, ifosfamide and paclitaxel (TIP) in mCRPC. Methods: We retrospectively analyzed patients with mCRPC treated with TIP at the University Hospital Hamburg-Eppendorf between November 2013 and September 2020. Accompanying in vitro analyses were performed using human prostate carcinoma cell lines harboring different levels of drug resistance including the docetaxel-resistant sublines PC3-DR and DU45-DR. Results: In total, 17 mCRPC patients treated with TIP were eligible for efficacy analyses with a median age of 65 yrs. At baseline, liver metastases were present in 88%, metastases of other visceral sides (lung, adrenal gland, brain) in 47% and bone metastases in 76% of the patients. Median hemoglobin was 9.8mg/dl, LDH 903 U/l and AP 205 U/l. Median PSA value was 77 ng/ml covering a wide range including three patients with a PSA-value below 1ng/ml. NSE was evaluated in 83% of the patients (median 38,5 U/l). Patients were extensively pretreated with a median of three treatment lines before TIP (100% docetaxel, 82% abirateron and/or enzalutamide, 47% cabazitaxel, 41% others). A median of 3,5 cycles of TIP were applied with 29% of the patients receiving the maximum of 6 cycles. Four patients discontinued treatment due to side effects (PNP, infection, ifosfamide induced psychosis). At interim analyses, 59 % of the patients showed a radiological response or stable disease with only one patient progressing till the end of treatment. Median PFS was 2.5 months, median OS 6 months. A decrease of PSA &gt; 30% and LDH &gt; 50% was observed in 41% and 35% of the patients, respectively. In vitro experiments revealed additive effects of TIP in 22Rv1, LNCaP and DU45 cells and synergistic effects in neuroendocrine LASCPC-01 cells. In PC3 cells, TIP induced antagonistic effects at lower doses, whereas dose-independent additive effects were observed in docetaxel-resistant PC3-DR. Surprisingly, preliminary data of combined therapies with different drug pairs suggest an antagonistic effect of paclitaxel in the combination with both, cisplatin and ifosfamide. Conclusions: Combinational therapy with cisplatin, ifosfamide and paclitaxel showed promising activity in some patients with aggressive mCRPC. Preclinical data suggest that the drug combination of cisplatin and ifosfamide rule the efficacy of TIP in mCRPC.

Fluorescent Products of Anthocyanidin and Anthocyanin Oxidation.

We found that the oxidation of cyanidin with hydrogen peroxide, tert-butyl hydroperoxide, 2,2'-azobis(2-methylpropionamidine), 3-morpholinosydnonimine hydrochloride, sodium hypochlorite, Fe3+, and Fe2+ induced the appearance of a new fluorescence band, centered at 525-540 nm when excited at 470-490 nm. The intensity of this fluorescence was related to the oxidant concentration. The same fluorescence was induced by the oxidation of other anthocyanidins, an anthocyanin (cyanidin-3-O-glucoside) as well as anthocyanin-rich red cabbage extract, and blackcurrant and elderberry juices. Peroxidized blood plasma also induced the appearance of cyanidin fluorescence. We also showed that the formation of the fluorescent product of pelargonidin can be observed in a culture of the MRC-5 human fetal lung fibroblast cell line and the DU-145 human prostate carcinoma cell line. Our results suggest that this new anthocyanidin/anthocyanin fluorescence may be an indicator of oxidation, especially of food products, where these compounds are present or added as colorants, and can also be useful to detect oxidation in biomedical experiments.

Does Bromelain-Cisplatin Combination Afford In-Vitro Synergistic Anticancer Effects on Human Prostatic Carcinoma Cell Line, PC3?

Background: Bromelain enhances anticancer impacts to chemotherapeutic agents. The question as to whether bromelain does promote in-vitro cytotoxic and proapoptotic effects of cisplatin on human prostatic carcinoma PC3 cell line was investigated. Materials and Methods: PC3 (human prostatic carcinoma) cells were treated either single or in combination with bromelain and/or cisplatin. MTT, clonogenic assay, flow cytometry and real-time quantitative polymerase chain reaction were used to investigate cell viability, colony formation, proapoptotic potential and p53 gene expression, respectively. Results: Cisplatin (IC10) combined with bromelain (IC40) significantly affected PC3 cell viability, inhibited colony formation, as well increased p53 proapoptotic gene expression compared to cisplatin single treatment. Nevertheless, bromelain-cisplatin chemoherbal combination did not display any additive proapoptotic effect compared to single treatments. Conclusion: Bromelain-cisplatin chemoherbal combination demonstrated synergistic in-vitro anticancer effect on human prostatic carcinoma cell line, PC3, that drastically reduced required cisplatin dose.

Does Bromelain-Cisplatin Combination Afford In-Vitro Synergistic Anticancer Effects on Human Prostatic Carcinoma Cell Line, PC3?

Background: Bromelain enhances anticancer impacts to chemotherapeutic agents. The question as to whether bromelain does promote in-vitro cytotoxic and proapoptotic effects of cisplatin on human prostatic carcinoma PC3 cell line was investigated. Materials and Methods: PC3 (human prostatic carcinoma) cells were treated either single or in combination with bromelain and/or cisplatin. MTT, clonogenic assay, flow cytometry and real-time quantitative polymerase chain reaction were used to investigate cell viability, colony formation, proapoptotic potential and p53 gene expression, respectively. Results: Cisplatin (IC10) combined with bromelain (IC40) significantly affected PC3 cell viability, inhibited colony formation, as well increased p53 proapoptotic gene expression compared to cisplatin single treatment. Nevertheless, bromelain-cisplatin chemoherbal combination did not display any additive proapoptotic effect compared to single treatments. Conclusion: Bromelain-cisplatin chemoherbal combination demonstrated synergistic in-vitro anticancer effect on human prostatic carcinoma cell line, PC3, that drastically reduced required cisplatin dose. [GMJ.2020;9:e1749]

Abstract 1634: Orthotopic and bone metastasis prostate cancer models using the 22Rv1 cell line

Abstract Prostate cancer is the most common cancer in men. Upon diagnosed malignancy the growth of the tumors is androgen driven in a majority of the cases. For that reason, the tumors are treated with androgen deprivation therapy (ADT) to stop or reduce tumor growth. Later, the tumors often become resistant to ADT and at this stage the disease is described as castration-resistant prostate cancer (CRPC). Bone metastasis is one of the hallmark of disease progression. Metastatic disease can be either androgen sensitive or castration resistant (mCRPC) in which the mortality rate is over 50%. Prostate cancer metastases are commonly formed in bones. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). The 22Rv1 cells are an invaluable model system to study AR function, the efficacy of existing drugs and to design novel anti-AR therapies that also target non-truncated regions of AR. The aim of this study was to establish predictive orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells were inoculated orthotopically into the prostate of NOG mice, and 0.5 × 106 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. 22Rv1 human prostate cancer cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% iFBS, 2 mM L-Glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/l D-Glucose and P/S. Tumor growth was followed by PSA measurements using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. For enhanced visualization of tumor models in 3D, we reconstructed the tissue from serial sections using a tailored computational pipeline based on applying a deformable model for corresponding points from adjacent sections. The observed tumor take rate was 100% in both models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. In conclusion, both presented models are suited to test the anticancer efficacy of new drug candidates. In addition, the intratibial model can be used to test the efficacy of novel drug candidates on prostate cancer cells in bone metastatic microenvironment. Citation Format: Justyna Zdrojewska, Tiina E. Kähkönen, Jenni H. Mäki-Jouppila, Masi Valkonen, Pekka Ruusuvuori, Jussi M. Halleen, Jenni Bernoulli. Orthotopic and bone metastasis prostate cancer models using the 22Rv1 cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1634.

Abstract 6377: Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates

Abstract It has been established that a cancer tissue contains, in general, a small subpopulation of cancer stem cells (CSCs) having tumor initiation capacity. Because CSCs are resistant to chemoradiotherapy and play important roles in cancer relapse and metastasis, strategies targeting CSCs are crucial for effective anticancer therapies. Curcumin (diferuloylmethane) isolated from the rhizome of the plant turmeric (Curcuma longa) is one of the most widely characterized phytochemicals and well known to suppress cancer cell proliferation, invasion, angiogenesis, and metastasis by regulating multiple cell signaling pathways. However, its bioavailability is limited due to poor absorption. To improve this disadvantage, Ohori et al. (Mol Cancer Ther 2006;5:2563-71.) synthesized a new curcumin analog GO-Y030 (1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-one), which has an about 30-fold greater growth-inhibitory effect than curcumin. In the current study, we verified the potency of GO-Y030 to act against CSC population. We first discovered that GO-Y030 could dose-dependently suppress a sphere-forming ability in human HuH7 hepatocellular carcinoma and human PC3 prostatic carcinoma cell lines. Interestingly, the specific inhibitor of the stress-inducible heat shock protein (HSP) 70 (also called HSP72, HSP70-1, or HSPA1A) exhibited effects similar to GO-Y030, i.e., inhibition of CSC sphere formation and upregulation of HSP70 and HSP40 protein expression. As this result suggested that target molecules of GO-Y030 might be HSP70 and its co-chaperones such as HSP40, we next performed in vitro HSP70/HSP40-mediated refolding activity assay and revealed that GO-Y030 efficiently inhibited HSP70/HSP40 chaperone activity. Finally, performing the substrate-binding assay, we have proven that GO-Y030 reduces the substrate-recognition and binding activity of HSP70. HSPs are molecular chaperones that function to prevent denaturation or unfolding of client proteins when they are threatened by stress or high temperature. Since CSCs by nature adapt to oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress by enhancing synthesis of protein-folding chaperones, the function of HSP70/HSP40 is important for the maintenance of CSC population. Our data suggest that GO-Y030 should impair stress tolerance in CSCs by inhibiting the interaction of HSP70 with substrate proteins and disrupting the function of HSP70/HSP40 and thus should finally contribute to reduction of CSC population. Citation Format: Maya Suzuki, Yohei Yamamoto, Aki Nishijima, Hiroyuki Shibata, Yasufumi Omori. Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6377.

GC-MS analysis, antimicrobial, antioxidant, antilipoxygenase and cytotoxic activities of Jacaranda mimosifolia methanol leaf extracts and fractions.

Jacaranda mimosifolia trees are grown in frost-free regions globally. The aim of this study was to evaluate the methanol crude extract and various fractions of increasing polarity of J. mimosifolia leaves for bioactive metabolites, as well as antimicrobial, antioxidant and anticancer activities. The anti-inflammatory potential of the various fractions of J. mimosifolia leaf extract was studied via the lipoxygenase (LOX) inhibitory assay. Methanol crude extract (ME), derived fractions extracted with chloroform (CF) and ethyl acetate (EAF), and residual aqueous extract (AE) of dried J. mimosifolia leaves were assayed for polyphenolic compounds, their antioxidant, antimicrobial and lipoxygenase (LOX) inhibitory activities, and anticancer properties. Polyphenolic compounds were determined via HPLC while phytochemicals (total phenolics, flavonoids, tannins and ortho-diphenol contents), antioxidant activities (DPPH, hydrogen peroxideperoxide, hydroxyl and superoxide radical anions) and LOX were measured via spectrophotometry. Methanol extracts and various fractions were evaluated for antibacterial activities against Bacillus subtilis, Klebsiella pneumonia, Pseudomonas aeruginosa and Staphylococcus aureus. Antifungal potential of the fractions was tested against three species: Aspergillus flavus, Aspergillus fumigatus and Fusarium oxysporum. The highest values for total phenolic content (TPC), total flavonoid content (TFC), flavonols, tannins and ortho-diphenols were in the ME, followed by CF > EAF > AE. ME also had the highest antioxidant activity with EC50 values 48±1.3, 45±2.4, 42±1.3 and 46±1.3 μg/mL based on the DPPH, hydrogen peroxide, hydroxyl radical and superoxide radical assays, respectively. TPC and TFC showed a significant, strong and positive correlation with the values for each of these antioxidant activities. ME exhibited anti-inflammatory potential based on its LOX inhibitory activity (IC50 = 1.3 μg/mL). ME also had the maximum antibacterial and antifungal potential, followed by EAF > CF > AE. Furthermore, ME showed the strongest cytotoxic effect (EC50 = 10.7 and 17.3 μg/mL) against human hormone-dependent prostate carcinoma (LnCaP) and human lung carcinoma (LU-1) cell lines, respectively. Bioactive compounds present in leaf methanol extracts of J. mimosifolia were identified using gas chromatography–mass spectrometry (GC–MS). Fifteen compounds were identified including phenolic and alcoholic compounds, as well as fatty acids. Our results suggest that J. mimosifolia leaves are a good source of natural products with antioxidant, anti-inflammatory and anti-cancer properties for potential therapeutic, nutraceutical and functional food applications.

Anti-prostate cancer activity of a nanoformulation of the spleen tyrosine kinase (SYK) inhibitor C61.

Patients with advanced or metastatic castration-resistant prostate cancer have a dismal prognosis and are therefore in urgent need for therapeutic innovations. Spleen tyrosine kinase has emerged as a new molecular target for castration-resistant prostate cancer. This study was done to test the cytotoxicity of the lead nanoformulation of a potent spleen tyrosine kinase inhibitor, C61-LNP, against the human prostatic carcinoma cell line, PC-3. PC-3 cells were treated with various concentrations of C61-LNP either alone or in combination with cisplatin (CDDP) for 24, 48 and 72 hours. The cell viability was evaluated by MTS assay. Cellular expression levels of various regulatory proteins in treated PC-3 cells were evaluated by Western blot analyses. C61-LNP exhibited dose-dependent cytotoxicity against PC-3 cells. C61-LNP, as well as C61-LNP + CDDP treatments, caused pro-apoptotic proteomic changes including an increase in cleaved fragments of caspases-3 and -9 consistent with caspase activation as well as an improvement in the anti-apoptotic Bcl2 and Bax levels. The combination of C61-LNP and CDDP changed in alterations of the cell cycle regulatory proteins p53, p21, p27, cyclin D1 and cyclin E levels. C61-LNP exhibited cytotoxicity against the castration-resistant prostate cancer cell line PC3. It also caused alterations in expression levels of regulatory proteins involved in apoptosis and cell cycle regulation and these effects were not abrogated by the standard chemotherapy drug CDDP. We are planning to further develop C61-LNP as a selective spleen tyrosine kinase inhibitor as part of a multi-modality treatment strategy for advanced/metastatic castration-resistant prostate cancer.

Biological Synthesis of CdTe Quantum Dots and Their Anti-Proliferative Assessment Against Prostate Cancer Cell Line.

Quantum dots (QDs) are semiconducting materials which have a wide array of applications starting from semiconducting devices, in humidity and pressure sensors and in medical imaging including cancer therapy. In the present study, cadmium telluride (CdTe) QDs were synthesized by a biological method using yeast cells, Saccharomyces cerevisiae in modified Czapek's medium. QDs were characterized by transmission electron microscopy and X-ray diffraction. Cancer cells were treated with 2, 4, 8 and 16 μM concentrations of CdTe QDs for 24 h. The anti-proliferative activity was determined by using MTT assay, by evaluating the production of reactive oxygen species (ROS), and also by nuclear apoptosis and cell cycle analysis using a flow cytometer against human prostate carcinoma cell line PC-3. The size of the CdTe QDs was approximately 2 nm. In vitro anti-proliferative study showed that CdTe QDs induced cell death and nuclear apoptosis in a dosedependent manner. CdTe QDs induced significant increase in ROS level in PC-3 cells which was dose-dependent. Moreover, CdTe also arrested growth of PC-3 cells in the G2/M phase of the cell cycle. This study elucidates the apoptotic activity of CdTe QDs on prostate carcinoma which could provide useful insights to researchers for its clinical application.

Chickpea peel waste as sustainable precursor for synthesis of fluorescent carbon nanotubes for bioimaging application

In this study, we report a controlled one-pot green synthesis of multiwalled carbon nanotubes (MWCNTs) via pyrolysis of sustainable agriculture waste (chickpea peel) at 400 °C in aqueous medium. These MWCNTs demonstrated 7.0 nm diameter, 0.28 nm graphitic spacing with carbonyl, hydroxyl, and carboxylic acid functionality. The D band (presence of sp3 defects) and G band (E2g mode of graphite) at 1350 cm−1 and 1580 cm−1 originated in Raman spectrum, respectively. The prepared MWCNTs showed blue fluorescence with 10% fluorescence quantum yield in aqueous medium. The MWCNTs showed triple exponential decay characteristics with an average fluorescence lifetime of 4.7 ns. The synthesized MWCNTs revealed a consistent fluorescence in the cytoplasm of 22RV1 human prostate carcinoma cell line without exerting any sign of cytotoxicity. The MWCNTs also exhibited remarkable cytocompatibility in human immortalized prostate epithelial RWPE1 cells.