Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy

Background

Non-small cell lung cancer (NSCLC) patients with L858R or exon 19 deletion mutations in epidermal growth factor receptor (EGFR) have good responses to the tyrosine kinase inhibitor (TKI), gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are resistant to gefitinib treatment. Here, we showed that curcumin can improve the efficiency of gefitinib in the resistant NSCLC cells both in vitro and in vivo models.

Methods/Principal Findings

After screening 598 herbal and natural compounds, we found curcumin could inhibit cell proliferation in different gefitinib-resistant NSCLC cell lines; concentration-dependently down-regulate EGFR phosphorylation through promoting EGFR degradation in NSCLC cell lines with wild-type EGFR or T790M EGFR. In addition, the anti-tumor activity of gefitinib was potentiated via curcumin through blocking EGFR activation and inducing apoptosis in gefitinib-resistant NSCLC cell lines; also the combined treatment with curcumin and gefitinib exhibited significant inhibition in the CL1-5, A549 and H1975 xenografts tumor growth in SCID mice through reducing EGFR, c-MET, cyclin D1 expression, and inducing apoptosis activation through caspases-8, 9 and PARP. Interestingly, we observed that the combined treatment group represented better survival rate and less intestinal mucosal damage compare to gefitinib-alone therapy. We showed that curcumin attenuated the gefitinib-induced cell proliferation inhibition and apoptosis through altering p38 mitogen-activated protein kinase (MAPK) activation in intestinal epithelia cell.

Conclusions/Significance

Curcumin potentiates antitumor activity of gefitinib in cell lines and xenograft mice model of NSCLC through inhibition of proliferation, EGFR phosphorylation, and induction EGFR ubiquitination and apoptosis. In addition, curcumin attenuates gefitinib-induced gastrointestinal adverse effects via altering p38 activation. These findings provide a novel treatment strategy that curcumin as an adjuvant to increase the spectrum of the usage of gefitinib and overcome the gefitinib inefficiency in NSCLC patients.     

Epidermal growth factor receptor inhibitor cancer drug gefitinib modulates cell growth and differentiation of acute myeloid leukemia cells via histamine receptors

BackgroundEpidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) is used for treating non-small cell lung cancer. Gefitinib also induces differentiation in acute myeloid leukemia (AML) cell lines and patient samples lacking EGFR by an unknown mechanism. Here we dissected the mechanism of gefitinib action responsible for its EGFR-independent effects.MethodsSignaling events were analyzed by homogenous time-resolved fluorescence and immunoblotting. Cellular proliferation and differentiation were assessed by ATP measurement, trypan blue exclusion, 5-bromo-2′-deoxyuridine incorporation and flow-cytometry. Gefitinib and G protein-coupled receptor (GPCR) interactions were assessed by β-arrestin recruitment, luciferase and radioligand competition assays. Role of histamine receptors (HR) in gefitinib actions were assessed by HR knockdown or pharmacological modulation. EGFR and HR interaction was assessed by co-immunoprecipitation.ResultsGefitinib reduced cyclic AMP content in both AML and EGFR-expressing cells and induced ERK phosphorylation in AML cells. Dibutyryl-cAMP or PD98059 suppressed gefitinib-induced AML cell cytostasis and differentiation. Gefitinib bound to and modulated HRs with subtype selectivity. Pharmacological or genetic modulations of H2 and H4 HRs (H2R and H4R) not only suppressed gefitinib-induced cytostasis and differentiation of AML cells but also blocked EGFR and ERK1/2 inhibition in MDA-MB-231 cells. Moreover, in MDA-MB-231 cells gefitinib enhanced EGFR interaction with H4R that was blocked by H4R agonist 4-methyl histamine (4MH).ConclusionHRs play critical roles in anti-cancer effects of gefitinib in both EGFR-deficient and EGFR-rich environments.General significanceWe furnish fresh insights into gefitinib functions which may provide new molecular clues to its efficacy and safety issues.     

Effect of an epidermal growth factor receptor inhibitor in mouse models of lung cancer

Gefitinib (Iressa, ZD1839) is a potent high-affinity competitive tyrosine kinase inhibitor aimed primarily at epidermal growth factor receptor (EGFR). Inhibitors in this class have recently been approved for clinical use in the treatment of advanced non-small cell lung cancer as monotherapy following failure of chemotherapy. We examined the efficacy of gefitinib on lung tumorigenesis in mouse models using both postinitiation and progression protocols. Gefitinib was given at a dose of 200 mg/kg body weight (i.g.) beginning either 2 or 12 weeks following carcinogen initiation. In the postinitiation protocol, gefitinib significantly inhibited both tumor multiplicity (approximately 70%) and tumor load (approximately 90%) in A/J or p53-mutant mice (P < 0.0001). Interestingly, gefitinib was also highly effective against lung carcinogenesis in the progression protocol when individual animals already have multiple preinvasive lesions in the lung. Gefitinib exhibited approximately 60% inhibition of tumor multiplicity and approximately 80% inhibition of tumor load when compared with control mice (both P < 0.0001). These data show that gefitinib is a potent chemopreventive agent in both wild-type and p53-mutant mice and that a delayed administration was still highly effective. Analyses of mutations in the EGFR and K-ras genes in lung tumors from either control or treatment groups showed no mutations in EGFR and consistent mutation in K-ras. Using an oligonucleotide array on control and gefitinib-treated lesions showed that gefitinib treatment failed to alter the activity or the expression level of EGFR. In contrast, gefitinib treatment significantly altered the expression of a series of genes involved in cell cycle, cell proliferation, cell transformation, angiogenesis, DNA synthesis, cell migration, immune responses, and apoptosis. Thus, gefitinib showed highly promising chemopreventive and chemotherapeutic activity in this mouse model of lung carcinogenesis.     

Allosteric modulation by protein kinase Cε leads to modified responses of EGF receptor towards tyrosine kinase inhibitors

Recently, we described a novel function of over-expressed protein kinase Cε (PKCε) as a negative allosteric modulator of EGFR signalling in several head and neck squamous carcinoma (HNSCC) cell lines. Extending this work, here we present several lines of evidence for the potency of PKCε to differently modulate the efficacy of EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and lapatinib. Using the HNSCC cell line FaDu as a model, we demonstrate by co-immunoprecipitation the physical association of over-expressed PKCε with the EGFR which is stabilised by gefitinib and leads to an increase in gefitinib-induced inhibition of EGFR downstream signalling and elevated EGFR-ErbB2 heterodimerisation. Cell cycle and Western blot analysis revealed that the gefitinib-induced apoptosis was enhanced whereas the pro-apoptotic effect of lapatinib that requires another EGFR conformation was reduced by PKCε. Our findings suggest that due to elevated expression PKCε may associate with the EGFR resulting in conformational changes and different allosteric modulation of the EGFR behaviour towards TKIs. This surprising capacity indicates PKCε as a novel predictive marker protein in molecular cancer therapy with EGFR tyrosine kinase inhibitors.     

The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, IressaR) and benzo[a]pyrene in human lung cancer

Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) affects MAPKs transduction pathways. Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[a]P co-treatment. Gefitinib enhances B[a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[a]P- and gefitinib-induced cytotoxicity, and B[a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.     

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa^®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.     

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.     

Epidermal growth factor receptor signaling modulates apoptosis via p38alpha MAPK-dependent activation of Bax in intestinal epithelial cells

Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38alpha. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38alpha phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38alpha-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss.     

Synergistic inhibitory effects by the combination of gefitinib and genistein on NSCLC with acquired drug-resistance in vitro and in vivo

In clinical practice, most patients with non small cell lung cancer (NSCLC) who respond to tyrosine kinase inhibitors eventually progress because of an acquired resistance mutation, T790M, in epidermal growth factor receptor (EGFR). Thus, it is important to identify a new drug to reduce resistance. The aim of this study was to test whether genistein combined with gefitinib is effective against NSCLC in a cell line carrying T790M, and to clarify the underlying mechanisms. The human lung cancer cell line H1975 was used as an in vitro and in vivo model. Cells were treated with gefitinib, genistein, or a combination at a range of concentrations. Cell proliferation was calculated to assess the anticancer effects of the compounds in vitro. Flow cytometry and Western blotting were employed to determine the inhibitory effects on proliferation and the induction of apoptosis. The in vivo effects of the compounds were examined using a xenografted nude mouse model for validation. Gefitinib together with genistein enhanced both growth inhibition and apoptosis; however, the greatest synergistic effect was observed at low concentrations. p-EGFR, p-Akt, and p-mTOR expressions in vitro were reduced more by the combined use of the drugs, whereas caspase-3 and PARP activities were increased. Significantly more tumor growth inhibition was detected following combination treatment in the in vivo model. These findings suggest that genistein enhanced the antitumor effects of gefitinib in a NSCLC cell line carrying the T790M mutation. This synergistic activity may be due to increased inhibition of the downstream molecular and pro-apoptotic effects of EGFR.     

BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations

Background

Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.

Methods and Findings

To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.

Conclusions

Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.     

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.     

ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma

The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, 'Iressa') blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dose-dependent growth arrest at G0-G1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.     

Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.