Chemosensitivity of radioresistant cells in the multicellular spheroids of A549 lung adenocarcinoma

Background

The relapse of cancer after radiotherapy is a clinical knotty problem. Previous studies have demonstrated that the elevation of several factors is likely in some way to lead to the development of treatment tolerance, so it is necessary to further explore the problem of re-proliferated radioresistant cells to chemotherapeutic agents. In the present study, we aimed to investigate the chemosensitivity of radioresistant cells originated from the multicellular spheroids of A549 lung adenocarcinoma.

Methods

After irradiated with 25 Gy of 6 MV X-ray to A549 multicellular spheroids, whose 10th re-proliferated generations were employed as radioresistant cells, and the control groups were A549 parental cells and MCF7/VCR resistant cells. The chemo-sensitivity test was made by six kinds of chemotherapeutic drugs which were DDP, VDS, 5-Fu, HCP, MMC and ADM respectively, while verapamil (VPL) was used as the reversal agent. Then the treatment effect was evaluated by MTT assay, and the multidrug resistant gene expressions of mdr1 and MRP were measured by RT-PCR.

Results

Both A549 parental cells and A549 derived radioresistant cells were resistant to DDP, but sensitive to VDS, 5-Fu, HCP, MMC and ADM. The inhibitory rates of VPL to these two types of cell were 98% and 25% respectively (P < 0.001). In addition, without drugs added, the absorbance value (A value) of A549 parental cells was 2-folds higher than that of their radioresistant cells (P < 0.001). As to the MCF7/VCR cells, they were resistant to DDP and VDS, but slight sensitive to MMC, ADM, 5-Fu, and HCP with 80% of inhibitory rate to VPL. The subsequent RT-PCR demonstrated that the Mdr1/β2-MG and MRP/β2-MG of all A549 cells were about 0 and 0.7 respectively, and those of MCF7/VCR cells were 35 and 4.36.

Conclusion

The chemosensitivity of A549 radioresistant cells had not changed markedly, and the decreased sensitivity to VPL could not be explained by the gene expression of mdr1 and MRP. It is possible that the changes in the cell membrane and decreased proliferate ability might be attributed to the resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to radioresistant cells. Therefore, the new biological strategy needs to be developed to treat recurring radioresistant tumor in combination with chemotherapy.     

Pharmacodynamics of DT-IgG, a dual-targeting antibody against VEGF-EGFR, in tumor xenografted mice

Purpose

DT-IgG is a fully humanized dual-target therapeutic antibody being developed to simultaneously target epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), important signaling molecules for tumor growth. The antitumor pharmacodynamics (PD) of DT-IgG was studied in nude mice bearing human tumor xenografts with different EGFR and VEGF expressions and K-ras oncogene status and compared with bevacizumab, cetuximab and bevacizumab?+?cetuximab.

Methods

Mice bearing human oral squamous cell carcinoma (Tu212), lung adenocarcinoma (A549), or colon cancer (GEO) subcutaneous xenografts were administered with the antibodies intraperitoneally (i.p.), and tumor volumes were measured versus time. Nonlinear mixed effects modeling (NONMEM) was used to study drug potencies (IC₅₀) and variations in tumor growth.

Results

The PD models adequately described tumor responses for the antibody dose regimens. In vivo IC₅₀ values varied with EGFR and K-ras status. DT-IgG had a similar serum t _1/2 as cetuximab (~1.7 vs. 1.5?day), was more rapid than bevacizumab (~6?day), and had the largest apparent distribution volume (DT-IgG?>?cetuximab?>?bevacizumab). The efficacy of DT-IgG was comparable to bevacizumab despite lower serum concentrations, but was less than bevacizumab?+?cetuximab.

Conclusions

A lower IC₅₀ of DT-IgG partially compensated for lower serum concentrations than bevacizumab and cetuximab, but may require higher doses for comparable efficacy as the combination. The model adequately predicted variations of tumor response at the DT-IgG doses tested and could be used for targeting specific tumor efficacies for future testing.     

Pharmacokinetics of liposomal-encapsulated and un-encapsulated vincristine after injection of liposomal vincristine sulfate in beagle dogs

Purpose

Vincristine sulfate (VCR) is a potent and widely used anti-tumor drug. Encapsulating VCR with liposomes improves its therapeutic index. However, there is little known about the pharmacokinetic features of un-encapsulated VCR (UE-VCR) and encapsulated VCR (E-VCR).

Methods

Two groups of beagle dogs were intravenously administered a single 0.07 mg/kg dose of VCR liposomal injection (L-VCR) and VCR ordinary injection (I-VCR), respectively. The concentrations of UE-VCR, E-VCR and total VCR (T-VCR) were determined by separating UE-VCR and E-VCR, using solid-phase extraction and validated liquid chromatography-tandem mass spectrometry-based methods. Pharmacokinetic parameters were calculated, using the compartment model. The pharmacokinetic parameters of L-VCR and I-VCR were compared using a Student’s t test.

Results

After intravenous injection of L-VCR, the pharmacokinetic parameters of E-VCR were similar to those of T-VCR. The concentrations of UE-VCR were very low, and its AUC _0–72h was only 2.5 % that of T-VCR. Compared with I-VCR, plasma AUC of E-VCR increased, with significantly extended distribution t _1/2 and reduced distribution volume of the peripheral department. C_2 min and AUC _0–1h of plasma UE-VCR decreased, with a similar elimination t _1/2.

Conclusions

The increased therapeutic index of L-VCR is demonstrated by the pharmacokinetic features, higher exposure to E-VCR and lower peak concentration of UE-VCR, following intravenous injection.     

MiR-224 promotes the chemoresistance of human lung adenocarcinoma cells to cisplatin via regulating G1/S transition and apoptosis by targeting p21WAF1/CIP1

Background:

Increasing evidence has shown that microRNAs (miRNAs) can serve as oncogenes and tumour suppressors to participate in tumour development. However, the roles of miRNAs in chemoresistance of human lung adenocarcinoma (LA) remain largely undefined.

Methods:

On the basis of miRNA microarray data, miR-224 was identified as the most upregulated miRNA in cisplatin (DDP; cis-diamminedichloroplatinum II)-resistant A549 cells compared with parental A549 cells. The aim of our study was to investigate the roles of miR-224 in the formation of DDP-resistant phenotype of LA cells and its possible molecular mechanisms.

Results:

Here we showed that miR-224 could promote the in vitro and in vivo DDP resistance of LA cells via regulating G₁/S cell cycle transition and apoptosis. p21^WAF1/CIP1, a potent cyclin-dependent kinase inhibitor, was identified as the direct and functional target gene of miR-224. Overexpression of p21^WAF1/CIP1 could phenocopy the effect of miR-224 downregulation and silencing of p21^WAF1/CIP1 could partially reverse the effect of miR-224 downregulation on DDP resistance of DDP-resistant LA cells. In addition, miR-224 could affect the G₁/S transition of cell cycle and apoptosis in LA cells through the p21^WAF1/CIP1-pRb pathway and the intrinsic mitochondrial death pathway. Furthermore, miR-224 was found to be downregulated in DDP-responding LA tissues, and its expression was inversely correlated with p21^WAF1/CIP1. Multivariate analyses indicated that the status of miR-224 might be an independent prognostic factor for predicting the survival of LA patients.

Conclusions:

Our findings shed novel light on the roles of miR-224/p21^WAF1/CIP1 signalling in the DDP resistance of LA cells, and targeting it will be a potential strategic approach for reversing the DDP resistance in human LAs.     

Characterization of the in vitro activity of AZD3409, a novel prenyl transferase inhibitor

Purpose

AZD3409 is a novel DPTI that has potent activity against both FTase and GGTase-1. The in vitro inhibition profile of AZD3409 was characterized using three different cell lines: mouse embryogenic fibroblasts, transfected with H-Ras^V12 (MEF), A549 cells (Ki4B-Ras mutation) and MCF-7 cells (no Ras mutation).

Methods

Both cytotoxicity and levels of inhibition of farnesylation and geranylgeranylation were determined in different assays in relation to the concentration of AZD3409. Results were compared with those obtained with the first-generation FTase inhibitor lonafarnib or the GGTase-1 inhibitor GGTI-2147.

Results

The mean IC₅₀ for cytotoxicity of AZD3409 and lonafarnib was 510 and 15,200?nM in MEF cells, 10,600 and 2,740?nM in A549 cells and 6,170 and 9,490?nM in MCF7 cells, respectively. In these cells, the IC₅₀ for FTase activity of AZD3409 ranged from 3.0 to 14.2?nM and of lonafarnib from 0.26 to 31.3?nM. The inhibiting activity of AZD3409 and lonafarnib on general protein farnesylation was comparable with the specific farnesylation levels of HDJ-2. In vitro geranylgeranylation of Rap1a could be inhibited by GGTI-2147 in all three cell lines, but only in MCF-7 cells by AZD3409. These results are in agreement with the IC₅₀ values for GGTase-1 activity as the lowest IC₅₀ for AZD3409 was found in the MCF-7 cell line.

Conclusions

AZD3409 inhibits farnesylation to a higher extent than geranylgeranylation. Both inhibition of farnesylation and geranylgeranylation could not be correlated to the antiproliferative activity of the drug.     

Cytotoxic Effect of Freeze-Dried Extract of Ecballium elaterium Fruit on Gastric Adenocarcinoma (AGS) and Esophageal Squamous Cell Carcinoma (KYSE30) Cell Lines

Purpose

Ecballium elaterium (L.) A. Rich (Cucurbitaceae), also known as the “squirting cucumber,” is a wild medicinal plant found abundantly in Moghan, Ardabil province, Iran. This study was undertaken to examine possible cytotoxic effect of freeze-dried aqueous extract of E. elaterium fruit on cell lines of gastric and esophageal origin namely called AGS (human gastric carcinoma) and KYSE30 (human esophageal squamous cell carcinoma).

Methods

The aqueous extract of the fruits of E. elaterium was prepared and freeze-dried. AGS and KYSE30 cancer cell lines were treated by the extract and incubated for 24, 48, and 72 h. Cytotoxicity was examined by MTT assay. Ethidium bromide/acridine orange (EB/AO) staining was used for apoptotic cell detection. A DAPI staining method was used to analyze cell cycle by flow cytometry.

Results

The IC₅₀ values were 2.5, 0.7, and 0.7 μg/ml for AGS cell line after 24, 48, and 72 h, respectively. IC₅₀ values for KYSE30 cell line were 500, 150, and 125 μg/ml after 24, 48, and 72 h, respectively. The EB/AO staining showed an increase in apoptotic cells. Cell cycle analysis showed a significant increase in cell density at G2/M phase.

Conclusions

The results of the current study showed that the freeze-dried aqueous extract of E. elaterium fruit has a cytotoxic effect on gastric and esophageal cancer cell lines by means of apoptosis. The gastric cancer cells (AGS) showed a remarkably higher sensitivity. It seems that several compounds are possibly responsible for the cytotoxic effect of the extract.     

Rapamycin suppresses ROS-dependent apoptosis caused by selenomethionine in A549 lung carcinoma cells

Purpose

Although selenium compounds possess chemotherapeutic features by inducing apoptosis in cancer cells with trivial side effects on normal cells, the mechanisms underlying its anti-cancer activity are insufficiently understood at the present. In this study, we investigated the effects of rapamycin on apoptosis induced by seleno-L-methionine (SeMet) or selenite in A549 cells.

Methods

The effects of Se compounds, SeMet and selenite, on cell proliferation, apoptosis and its signaling pathway were investigated in established human adenocarcinoma cell line (A549). Cancer cells were treated with each Se during different periods. Cell apoptosis and signaling molecules were analyzed by flow cytometry (TUNEL method) or immunoblotting, respectively.

Results

SeMet induces reactive oxygen species generation associated with the induction of apoptosis, because pretreatment of cells with N-acetyl-L-cysteine completely blocked SeMet-induced apoptosis. We also found that rapamycin completely suppressed the apoptosis of cells treated by SeMet, but not selenite. SeMet-induced apoptosis is significantly downregulated in combination with PI3?K family inhibitors (LY294002, wortmannin, PI-103, and 3-methyladenine). In addition, ROS generation was included in downstream signaling events associated with the phosphorylation of mTOR, because pretreatment of cells with rapamycin inhibited ROS generation.

Conclusion

These results suggest that SeMet-induced apoptosis is affected by the Akt/mTOR/ROS pathway in A549 cells. Akt serves an anti-survival function in the system of SeMet-treated lung cancer cells, but autophagic signaling remained unsolved.     

托瑞米芬协同顺铂对人肺癌细胞株A549的影响

目的：研究托瑞米芬（TOR）对人肺腺癌细胞系A549的毒性作用及其与顺铂（DDP）联用的协同效应,探讨肺癌综合治疗的方向。方法：用MTT显色法检测TOR及与DDP联用后对A549细胞的毒性作用,测定其吸光度（A）值。用流式细胞仪检测细胞DNA含量,Western blot法检测p21蛋白表达。结果：TOR能直接抑制A549细胞的生长,≥5μmol/L的TOR可明显增强DDP的细胞毒性作用。TOR可加强DDP对S期、G2期及M期细胞的作用,且DDP＋TOR后p21蛋白表达增加。结论：≥5μmol/L的TOR与DDP联用对A549细胞具有显著的协同抗肿瘤效应。     

Mechanisms of enhanced tumoricidal efficacy of multiple small dosages of ranpirnase, the novel cytotoxic ribonuclease, on lung cancer

Purpose

The effect of multiple small dosages of the cytotoxic RNase, ranpirnase (ONCONASE^®, ONC), on lung cancer was studied. The possible mechanisms for the enhanced tumoricidal efficacy of multiple small dosages of ONC were also investigated.

Methods

Hematoxylin and eosin staining, TUNEL labeling, and caspase-3-antibody labeling were used for in vivo analysis of apoptosis. A growth-delay assay was applied to detect the therapeutic potential of small and multiple dosages of ONC in vivo. ONC-induced changes in blood flow in A549 tumors and the kidney were measured non-invasively by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI).

Results

In cell culture studies, ONC significantly inhibited tumor growth of A549 human NSCLC cells without damaging non-cancerous cells (HLF-1 human lung fibroblast). Multiple small dosages of ONC significantly prolonged tumor growth delay of A549 tumors, with increased apoptosis in vivo from 0.5 ± 0.3 to 70.1 ± 1.1% (by TUNEL labeling, N = 3, P < 0.05). Interestingly, multiple small doses of ONC were more effective than a single large dose for the inhibition of tumor growth with reduced side effect. Using non-invasive DCE-MRI methods, we found that the mean of the K ^trans median values increased to 49.3 ± 7.5% from the pre-ONC values by ONC (N = 4 mice, P < 0.05). A subsequent T ₁ map of the kidney showed that T ₁ values were temporarily decreased for up to 2 days (however, fully recovered ～4 days post-treatment).

Conclusions

Multiple small dosages of ONC significantly inhibited tumor growth of A549 NSCLC cells in vivo, with markedly increased apoptosis. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.     

Arsenic trioxide exerts synergistic effects with cisplatin on non-small cell lung cancer cells via apoptosis induction

Background

Despite multidisciplinary treatment, lung cancer remains a highly lethal disease due to poor response to chemotherapy. The identification of therapeutic agents with synergistic effects with traditional drugs is an alternative for lung cancer therapy. In this study, the synergistic effects of arsenic trioxide (As₂O₃) with cisplatin (DDP) on A549 and H460 non-small cell lung cancer (NSCLC) cells were explored.

Methods

A549 and H460 human lung cancer cells were treated with As₂O₃ and/or DDP. Cell growth curves, cell proliferation, cell cycle, and apoptosis of human cancer cell lines were determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, clonogenic assay, and flow cytometry (FCM). Apoptosis was further assessed by TUNEL staining. Cell cycle and apoptosis related protein p21, cyclin D1, Bcl-2, bax, clusterin, and caspase-3 were detected by western blot.

Results

MTT and clonogenic assay showed As₂O₃ within 10^-2 μM to 10 μM exerted inhibition on the proliferation of NSCLC cells, and 2.5 μM As₂O₃ exerted synergistic inhibition on proliferation with 3 μg/ml DDP. The combination indices (CI) for A549 and H460 were 0.5 and 0.6, respectively, as confirmed by the synergism of As₂O₃ with DDP. FCM showed As₂O₃ did not affect the cell cycle. The G0/G1 fraction ranged from 57% to 62% for controlled A549 cells and cells treated with As₂O₃ and/or DDP. The G0/G1 fraction ranged from 37% to 42% for controlled H460 cells and cells treated with As₂O₃ and/or DDP. FCM and TUNEL staining illustrated that the combination of As₂O₃ and DDP provoked synergistic effects on apoptosis induction based on the analysis of the apoptosis index. Western blotting revealed that the expression of cell cycle related protein p21 and cyclin D1 were not affected by the treatments, whereas apoptosis related protein bax, Bcl-2, and clusterin were significantly regulated by As₂O₃ and/or DDP treatments compared with controls. The expression of caspase-3 in cells treated with the combination of As₂O₃ and DDP did not differ from that in cells treated with a single agent.

Conclusion

As₂O₃ exerted synergistic effects with DDP on NSCLC cells, and the synergistic effects were partly due to the induction of caspase-independent apoptosis.     

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines

Background:

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity.

Methods:

Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT–PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays.

Results:

Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC₅₀, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers.

Conclusions:

Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.     

Pharmacokinetics of eribulin mesylate in patients with solid tumors and hepatic impairment

Purpose

The aim of this study was to determine the plasma pharmacokinetics of eribulin mesylate in patients with solid tumors with mild and moderate hepatic impairment.

Patients and methods

A phase I, pharmacokinetic study was performed in patients with advanced solid tumors and normal hepatic function or Child-Pugh A (mild) or Child-Pugh B (moderate) hepatic impairment. Treatments were given on day 1 and 8 of a 21-day cycle and consisted of 1.4, 1.1 and 0.7?mg/m² eribulin mesylate, for normal hepatic function, Child-Pugh A and B hepatic impairment, respectively. Also safety and anti-tumor activity were determined.

Results

Hepatic impairment increased exposure to eribulin. In patients with Child-Pugh A (N?=?7) and Child-Pugh B (N?=?5), mean dose-normalized AUC_0?C?? was 1.75-fold (90?% confidence intervals (CI): 1.16?C2.65) and 2.48-fold (90?% CI: 1.57?C3.92) increased, respectively, compared with patients who have normal function (N?=?6). The most frequently reported treatment-related events were alopecia (12/18) and fatigue (7/18) and these were observed across all groups. Nine patients (50?%) had stable disease as best response.

Conclusions

A reduced dose of 1.1 and 0.7?mg/m² of eribulin mesylate is recommended for patients with Child-Pugh A or B hepatic impairment, respectively.     

High expression of heme oxygenase-1 is associated with tumor invasiveness and poor clinical outcome in non-small cell lung cancer patients

Background

Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, is known to play a role in the protection of cells against oxidative stress, inflammation, anomalous proliferation and apoptosis. As yet, the role of HO-1 expression in non-small cell lung cancer (NSCLC) development and metastasis remains unclear and insufficient data are available regarding its impact on the prognosis of NSCLC patients.

Methods

Seventy NSCLC patients who underwent surgical resection were included in this HO-1 expression study and, concomitantly, clinical parameters were collected. Two lung adenocarcinoma cell lines (A549 and H441) were used to assess both invasive and migratory parameters in vitro.

Results

NSCLC patients with a high HO-1 expression ratio (tumor tissue/normal tissue) (> 1) exhibited a significantly poorer prognosis and a higher metastatic rate compared to those with a low HO-1 expression ratio (p?<?0.05). The invasive and migratory abilities of A549 and H441 cells significantly increased after exogenous HO-1 over-expression and significantly decreased after siRNA-mediated HO-1 expression silencing. HO-1 up- and down-regulation also positively correlated with the expression of metastasis-associated proteins EGFR, CD147 and MMP-9. In addition, we found that HO-1 expression can be inhibited by PI3K and AKT inhibitors, but not by MAPK inhibitors.

Conclusions

HO-1 is a poor prognostic NSCLC predictor and its over-expression may increase the metastatic potential of NSCLC. Based on our findings and those of others, HO-1 may be considered as a novel NSCLC therapeutic target.     

(−)-Epigallocatechin-3-gallate inhibits human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer cells by targeting HIF-1α

Purpose

To investigate the effects of (?)-epigallocatechin-3-gallate (EGCG) on human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer (NSCLC) cells and the underlying mechanisms.

Methods

NSCLC cells (A549 and NCI-H460) transfected with EGFP plasmids containing HPV-16 E6 or E7 oncogene were treated with different concentrations of EGCG for 16 h. The effects of EGCG on angiogenesis in vitro and in vivo were observed. The expression of HIF-1α, p-Akt, and p-ERK1/2 proteins in NSCLC cells was analyzed by Western blot. The levels of HIF-1α mRNA in NSCLC cells were detected by real-time RT-PCR. The concentration of VEGF and IL-8 in the conditioned media was determined by ELISA. HIF-1α, VEGF, and CD31 expression in A549 xenografted tumors of nude mice was analyzed by immunohistochemistry.

Results

HPV-16 E6 and E7 oncoproteins HIF-1α-dependently promoted angiogenesis in vitro and in vivo, which was inhibited by EGCG. Mechanistically, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression in NSCLC cells. Additionally, 50 and 100 μmol/L of EGCG significantly reduced the secretion of VEGF and IL-8 proteins induced by HPV-16 E7 oncoprotein in NSCLC A549 cells. Meanwhile, HPV-16 E6 and E7 oncoproteins HIF-1α-dependently enhanced Akt activation in A549 cells, which was suppressed by EGCG. Furthermore, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α and HIF-1α-dependent VEGF and CD31 expression in A549 xenografted tumors.

Conclusions

EGCG inhibited HPV-16 oncoprotein-induced angiogenesis conferred by NSCLC through the inhibition of HIF-1α protein expression and HIF-1α-dependent expression of VEGF, IL-8, and CD31 as well as activation of Akt, suggesting that HIF-1α may be a potential target of EGCG against HPV-related NSCLC angiogenesis.     

The marine sponge toxin agelasine B increases the intracellular Ca2+ concentration and induces apoptosis in human breast cancer cells (MCF-7)

Purpose

In search for new drugs derived from natural products for the possible treatment of cancer, we studied the action of agelasine B, a compound purified from a marine sponge Agelas clathrodes.

Methods

Agelasine B was purified from a marine sponge Agelas clathrodes and assayed for cytotoxicity by MTT on two human breast cancer cells (MCF-7 and SKBr3), on a prostate cancer cells (PC-3) and on human fibroblasts. Changes in the intracellular Ca²⁺ concentrations were assessed with FURA 2 and by confocal microscopy. Determination of Ca²⁺-ATPase activity was followed by Pi measurements. Changes in the mitochondria electrochemical potential was followed with Rhodamine 123. Apoptosis and DNA fragmentation were determined by TUNEL experiments.

Results

Upon agelasine B treatment, cell viability of both human breast cancer cell lines was one order of magnitude lower as compared with fibroblasts (IC₅₀ for MCF-7?=?2.99???M; SKBr3: IC₅₀?=?3.22???M vs. fibroblasts: IC₅₀?=?32.91???M), while the IC₅₀ for PC-3 IC₅₀?=?6.86???M. Agelasine B induced a large increase in the intracellular Ca²⁺ concentration in MCF-7, SKBr3, and PC-3 cells. By the use of confocal microscopy coupled to a perfusion system, we could observe that this toxin releases Ca²⁺ from the endoplasmic reticulum (ER). We also demonstrated that agelasine B produces a potent inhibition of the ER Ca²⁺-ATPase (SERCA), and that this compound induced the fragmentation of DNA. Accordingly, agelasine B reduced the expression of the anti-apoptotic protein Bcl-2 and was able to activate caspase 8, without affecting the activity of caspase 7.

Conclusions

Agelasine B in MCF-7 cells induce the activation of apoptosis in response to a sustained increase in the [Ca²⁺] _i after blocking the SERCA activity. The reproduction of the effects of agelasine B on cell viability and on the [Ca²⁺] _I obtained on SKBr3 and PC-3 cancer cells strongly suggests the generality of the mechanism of action of this toxin.     

Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models

Background

Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models.

Methods

A diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ.

Results

CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC₅₀ values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC₅₀ values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP).

Conclusions

CFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.

     

The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate,as compared to methotrexate,in HeLa cells in vitro

Purpose

To investigate the impact of 5-formyltetrahydrofolate on the activities of pralatrexate, as compared to methotrexate (MTX), in vitro.

Methods

Cells were exposed to (6S)5-formyltetrahydrofolate (5-formylTHF) for 24 h, before or after a 6-h exposure to antifolates following which the cellular accumulation and activities of the drugs were evaluated in HeLa cells.

Results

A 24-h delay between a 6-h exposure to antifolates and a subsequent 24-h exposure to 4 μM 5-formylTHF sustained the full activities of both antifolates. A 72-h interval was required between a single exposure of up to 4 μM 5-formylTHF and subsequent exposure to drugs to sustain activities of the antifolates. When cells were incubated with 4 μM 5-formylTHF for 24 h weekly, for 4 weeks, there was no significant increase in the IC₅₀ for pralatrexate, but the MTX IC₅₀ increased 2.5-fold as compared to cells growing continuously in 25 nM 5-formylTHF. This cyclical exposure to 5-formylTHF increased the cell folate pool by 16 %, had no significant effect on the intracellular pralatrexate level, but decreased intracellular MTX by 15 %. An extracellular concentration of MTX 50-fold higher than that of pralatrexate was required to achieve an intracellular level, and growth inhibition, comparable to that of pralatrexate.

Conclusions

Cyclical exposures to 5-formylTHF at levels in excess of what is achieved in most clinical “rescue” regimens do not affect pralatrexate accumulation nor antitumor activity in HeLa cells, in contrast to MTX. An important element in preserving pralatrexate activity is achieving a sufficient interval between exposure to 5-formylTHF and the next dose of antifolate.