Identification of ING4 (Inhibitor of Growth 4) as a Modulator of Docetaxel Sensitivity in Human Lung Adenocarcinoma

Resistance to docetaxel (DTX) usually occurs in patients with lung adenocarcinoma. To better elucidate the underlying molecular mechanisms involved in resistance to DTX-based chemotherapy, we established a DTX-resistant lung adenocarcinoma cell line (SPC-A1/DTX). By gene array analysis, the expression of ING4was found to be significantly downregulated in SPC-A1/DTX cells. Additionally, the decreased expression of the ING4 gene was induced upon DTX treatment of SPC-A1 cells. Overexpression of ING4 reverses DTX or paclitaxel resistance of DTX-resistant lung adenocarcinoma cells (SPC-A1/DTX or A549/Taxol) by inducing apoptosis enhancement and G 2 /M arrest, and small interfering RNA–mediated ING4knockdown renders DTX-sensitive lung adenocarcinoma cells more resistant to DTX or paclitaxel. Also, overexpression of ING4 could enhance the in vivo sensitivity of SPC-A1/DTX cells to DTX. The phenotypical changes of SPC-A1/DTX cells induced by overexpression of ING4might be associated with the decreased ratio of Bcl-2/Bax, which resulted in the activation of caspase-3. The level of ING4expression in tumors of nonresponding patients was significantly lower than that in those of responders, suggesting that the expression of ING4was positively correlated with tumor response to DTX. Our results provide the first evidence that ING4might be essential for DTX resistance in lung adenocarcinoma. Thus, ING4will be a potential molecular target for overcoming resistance to DTX-based chemotherapies in lung adenocarcinoma.


INTRODUCTION
Non-small cell lung cancer (NSCLC) has been one of the most lethal malignancies around the world. Adenocarcinoma of the lung is the most common type of lung cancer and accounts for 30% to 35% of primary lung tumors (1). Currently, systemic chemotherapy is still an important treatment option for patients with advanced lung adenocarcinoma (2). Docetaxel (DTX) has become an integral part of several commonly used chemotherapy regimens in NSCLC (3). However, the development of intrinsic or acquired resistance to DTX remains the greatest obstacle to the successful treatment of patients with lung adenocarcinoma. Thus, elucidating the mechanisms by which DTX resistance arises in lung adenocarcinoma remains a critical issue for overcoming and predicting DTX resistance in NSCLC.
Recently, a wealth of novel insights into molecular targets and mechanisms of cancer chemosensitivity and resistance have yielded much progress in genomic and proteomic studies (4). DTX (Taxotere TM ) is a novel microtubule-stabilizing agent that has been synthesized from a precursor extracted from a renewable natural source, the needles of the European yew, Taxus baccata (5). This agent can enhance microtubule assembly and inhibit the depolymerization of tubulin. In the clinical setting, DTX has been used for adjuvant therapy after resection of localized NSCLC and in combination with radiation for locally advanced NSCLC and treatment of patients with advanced NSCLC (6,7). However, the therapeutic results in some patients with advanced NSCLC have been unsatisfying, as in cases of intrinsic or acquired chemoresistance. The dysregulation of oncogenes such as Bcl-2 family members or tumor suppressors such as PUMA (p53 upregulated modulator of apoptosis) has been found to be associated with DTX resistance of tumor cells (8)(9)(10). However, the molecular mechanisms of DTX resistance are very complicated and require further elucidation.
Previously, we successfully established a DTX-resistant lung adenocarcinoma cell line (SPC-A1/DTX) from a DTXnonresistant lung adenocarcinoma cell

Identification of ING4 (Inhibitor of Growth 4) as a Modulator of Docetaxel Sensitivity in Human Lung Adenocarcinoma
line (SPC-A1). To gain further insight into the mechanisms of DTX resistance and explore novel potential therapeutic targets for reversing the DTX resistance of lung adenocarcinoma, we performed a microarray analysis on lung adenocarcinoma cells using the Affymetrix U133A microarray, which showed that a total of 2332 genes that were differentially expressed between the SPC-A1 and SPC-A1/DTX cell lines. Among these genes, ING4 (inhibitor of growth 4) was found to be significantly downregulated in the SPC-A1/DTX cell line in comparison with the parental SPC-A1 cell line. ING4, a novel member of the inhibitor of growth (ING) gene family, has attracted much attention as a tumor suppressor because of its ability to suppress tumor growth, angiogenesis and invasion (11). In our previous studies, we also showed that downregulation of ING4 was associated with poor prognosis of patients with lung adenocarcinoma (data not published). However, there have been no reports about the association of ING4 expression with DTX sensitivity of lung adenocarcinoma.
In the present study, we attempted to investigate the roles of ING4 in docetaxel-induced drug resistance and its possible molecular mechanisms. Herein, we report our finding that restoration of ING4 expression could reverse the resistance of NSCLC cells to DTX both in vitro and in vivo by inducing apoptosis enhancement and cell cycle G 2 /M arrest. Also, the expression of ING4 in advanced lung adenocarcinoma might be positively correlated with the response of patients to DTX. Taken together, our results indicate that ING4 might be a key regulator of DTX resistance in lung adenocarcinoma cells and has the potential of being a therapeutic target for chemosenstization of lung adenocarcinoma.

Cell Lines and Chemotherapeutic Reagents
The human lung adenocarcinoma cell lines (SPC-A1 or A549) and taxolresistant human lung adenocarcinoma cell line (A549/Taxol) were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). The DTXresistant lung adenocarcinoma cell line (SPC-A1/DTX) was established and preserved in our lab. The DTX-resistant SPC-A1 cell line was selected by continuous exposure to increasing concentrations of DTX. DTX was added into exponentially growing cultures of SPC-A1 cells at a concentration of 0.008 μg/L and allowed to remain in the culture until cell growth resumed. The cultures were then split and treated again with progressively higher concentrations of DTX. Over the course of selection, the DTX concentration was increased to 5.0 μg/L. The resulting subline was designated as SPC-A1/DTX cells (SPC-A1/DTX). The taxol-resistant A549 cell line (A549/ Taxol) was preserved in a 0.2-μg/mL final concentration of taxol according to the manufacturer's instruction. All cell lines were cultured in RPMI 1640 (GIBCO-BRL, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin in humidified air at 37°C with 5% CO 2 . DTX and paclitaxel and pancaspase inhibitor (Z-VAD-FMK) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Stock solutions of DTX and paclitaxel (1.0 μg/mL) were prepared with dimethyl sulfoxide and diluted with phosphate-buffered saline (PBS) to the required concentrations before each experiment.

Microarray Analysis
Total RNA from the lung adenocarcinoma cell line (SPC-A1) or the corresponding DTX-resistant lung adenocarcinoma cell line (SPC-A1/DTX) was isolated by use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quality and quantity of the RNA samples were assessed by standard electrophoresis and spectrophotometry methods. Complementary DNA (cDNA) microarray analysis was performed with reagents and according to protocols provided by Affymetrix (Santa Clara, CA, USA). Briefly, we prepared double-stranded cDNA using the One-Cycle cDNA synthesis kit. The GeneChip IVT labeling kit was then used to synthesize biotinlabeled cRNA, which was then fragmented prior to hybridization. The labeled, fragmented cRNA samples were then hybridized to Affymetrix U133A microarrays, and the array was washed, stained and scanned with the Affymetrix GeneChip Scanner 3000. The acquired image was analyzed by the Affymetrix GeneChip operating software version 1.0. Differential cDNA expression was determined with a two-sided t test on a single cDNA basis. Differentially detected signals were generally accepted as true when the ratio of the p value was < 0.05 and were then selected for cluster analysis.

Real-Time Reverse Transcription-Polymerase Chain Reaction Assay
Total cellular RNA was prepared using TRIzol reagent (Invitrogen) and reversely transcribed according to the manufacturer's instruction. Real-time polymerase chain reaction (PCR) products were detected with SYBR Green I dye by using a Light Cycler instrument (Roche, Basel, Switzerland). The GAPDH gene was amplified as an internal control. Relative quantitation was done by using the ΔCt (threshold cycle) method by taking the difference (ΔCt) between the Ct of GAPDH and ΔCt of each transcript and computing 2 -ΔCt . We obtained the sequences of primers for ING4 by referring to Tzouvelekis et al. (12). The sequences of primers for the ING4 gene were as follows: sense 5′-AGCTT GCCAT GCAGA CCT-5′; reverse 5′-GCGCA CGAGC TTTAA CTT-3′. Ct values were normalized to the reference gene GAPDH. The sequences of primers for the GAPDH gene (sense 5′-GAAGG TGAAG GTCGG ATGC-3′; reverse 5′-GAAGA TGGTG ATGGG ATTTC-3′) were designed by using the Primer Premier 5.0 software package.

Transfection and Stable Selection
The parental SPC-A1 or A549 and resistant SPC-A1/DTX or A549/Taxol cells were seeded into 6-well plates at 2.0 × 10 4 cells/well, respectively, and cultured overnight to 80% confluence prior to transfection. We performed transfection

Flow Cytometric Analysis of Apoptosis
An annexin V-fluorescein isothiocyanate apoptosis detection kit (Oncogene Research Products, Boston, MA, USA) was used to detect apoptosis according to the manufacturer's instructions.

Flow Cytometric Analysis of Cell Cycle
Cells were harvested at the 70% confluent stage and fixed in 70% ethanol at -20°C. After being washed with PBS, the cells were treated with PBS containing RNase A (100 mg/mL) at 37°C for 30 min. After centrifugation, the cells were resuspended in PBS containing propidium iodide (50 μg/mL) and stained at room temperature for 30 min. DNA content was evaluated by use of a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and CellQuest software (Becton Dickinson).

Assay of Caspase-3 Activity
Caspase-3 activity was determined by use of the colorimetric CaspACE Assay System (Promega Corp., Madison, WI, USA) following the manufacturer's instructions, as previously reported (14). Each determination was performed in triplicate.

In Vivo Chemosensitivity Assay
Animal studies were performed according to institutional guidelines. Approximately 5.0 × 10 6 SPC-A1/DTX/ control or SPC-A1/DTX/ING4 cells were suspended in 100 μL PBS and injected subcutaneously into the right side of the posterior flank of female BALB/c athymic nude mice (Department of Comparative Medicine, Jinling Hospital, Nanjing, China) at 5 to 6 wks of age. Tumor growth was examined every other day with a vernier caliper. Tumor volumes were calculated by using the equation: , wherein A is the largest diameter, and B is the perpendicular diameter. When the average tumor size reached about 50 mm 3 , DTX was given through intraperitoneal injection with a concentration of 1.0 mg/kg, 1 dose every other day with 3 doses totally. After 2 wks, all mice were killed, and necropsies were performed. The primary tumors were excised, paraffin embedded and formalin fixed. Then we performed hematoxylin and eosin staining and immunostaining analysis for PCNA protein expression and analyzed the apoptosis with a TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) apoptosis detection kit (KeyGEN, Nanjing, China) according to the manufacturer's instructions.

Patients and Tissue Samples
A total of 18 lung adenocarcinoma tissues were collected from patients with advanced lung adenocarcinoma who received chemotherapy at the Department of Medical Oncology, Jinling Hospital, between March 2005 and September 2006. Patients met all of the following criteria: suffering from primary lung adenocarcinoma; a histological diagnosis of lung adenocarcinoma with at least one measurable lesion; a clinical stage of IIIB-IV; first-line chemotherapy either with DTX 75 mg/m 2 and cisplatin 100 mg/m 2 or DTX 75 mg/m 2 and carboplatin AUC (area under the curve) 6 mg/mL/min administered every 3 wks for a maximum of 5 cycles; and availability of sufficient tumor tissue in paraffin blocks for assessment by immunohistochemistry. Tumor response was examined by computed to-mography and evaluated according to the Response Evaluation Criteria in Solid tumors (RECIST) as a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD), as described previously (16). Written permission to use human tumor tissues was obtained from the patients.

Statistical Analysis
Experimental data were expressed as the mean ± SD of at least three independent assays. Statistical analyses were carried out using one-way ANOVA and Student t test to evaluate the continuous variables. Progression-free survival (PFS) was assessed from the first day of chemotherapy administration to the date of objective disease progression. The probability of survival was plotted by the Kaplan-Meier method and compared by the log-rank test. Differences between groups were considered significant at p < 0.05. All statistical analyses were per-formed using the SPSS 13.0 statistical software.
All supplementary materials are available online at www.molmed.org.

Analysis of Growth or Cytotoxicity of DTX in DTX-Nonresistant or -Resistant Lung Adenocarcinoma Cells
The DTX-resistant cell line (SPC-A1/ DTX), was developed from the DTXnonresistant cell line (SPC-A1). As observed by optical microscopy (Figure 1A), the morphology of the SPC-A1 and SPC-A1/ DTX cells was significantly different and the SPC-A1/DTX cells appeared as large swellings or spindle-or rhombus-shaped cell forms. The doubling time of the SPC-A1/DTX cell line (34.0 h) was significantly longer than that of the SPC-A1 cell line (26.5 h) (p < 0.05; Figure 1B). With the relative resistance (as a resistance factor) calculated via the ratio of the half maximal inhibitory concentration (IC 50 )-resistant variant/IC 50 of the parental cell line, the SPCA-1/DTX cell line was 5.37-fold resistant to DTX (13.58 μg/L) and was 3.46-fold resistant to paclitaxel (0.61 μg/L) ( Figure 1C), suggesting that SPC-A1/DTX cell line acquired resistance to cross-resistant to paclitaxel. Colony formation assays also showed significant DTX resistance in the SPC-A1/ DTX compared with the SPC-A1 cell line ( Figure 1D). Therefore, the sensitivity of DTX was shown to be significantly different between the SPC-A1 and SPC-A1/ DTX cell lines, a finding that might provide us better cell models to investigate the molecular mechanisms of DTX resistance in lung adenocarcinoma.

DTX Resistance of Lung Adenocarcinoma Cells was Correlated with Loss of the G 2 /M Checkpoint in the Cell Cycle
To investigate whether resistance to DTX was correlated with drug-induced apoptosis and/or cell-cycle distribution, we performed a flow cytometry assay to detect the changes in apoptosis and cell cycles. In the SPC-A1/DTX cell line, there was a significant decrease in apoptosis on exposure to various doses of DTX in comparison with the parental SPC-A1 cell line (Figure 2A). Likewise, following treatment with various doses of paclitaxel, a significant reduction in the extent of cell death was also seen by comparison of parental SPC-A1 and SPC-A1/DTX cells. Next, the perturbation in cell cycle following exposure to DTX (1.0 μg/L) was analyzed. After 24-h exposure to DTX in parent SPC-A1 cells, the percentage of G 2 /M-phase cells was significantly increased (p < 0.05), and an apoptosis peak (sub-G1) could be seen before the G 1 phase. However, in DTX-resistant SPC-A1/ DTX cells, there were no obvious changes in the sub-G1 population. Furthermore, the percentage of G 2 /M-phase cells was obviously decreased in resistant SPC-A1/DTX cells relative to parental SPC-A1 cells (p < 0.05; Figure 2B). Therefore, it was concluded that loss of the G 2 /M cell-cycle checkpoint function and abrogation of apoptosis might be involved in the acquired DTX resistance of lung adenocarcinoma cells.

Microarray Analysis of Differentially Expressed Genes Associated with DTX Resistance of Lung Adenocarcinoma Cells
To investigate the molecular mechanisms of DTX resistance in lung adenocarcinoma cells, we performed a microarray analysis on lung adenocarcinoma cells using the Affymetrix U133A microarray. The microarray data showed that a total of 2332 genes were differentially expressed between the SPC-A1 and SPC-A1/DTX cell lines (Supplementary Figure S1A; p < 0.05). By the fold-change analysis, we found that 338 or 31 of the 29,187 flagged cDNAs in SPC-A1/DTX cells showed at least a 4.0-or 15.0-fold change in expression level compared with parental SPC-A1 cells. Compared with SPC-A1 cells, 12 genes were significantly downregulated in SPC-A1/DTX cells, whereas the other 19 genes were significantly upregulated in SPC-A1/ DTX cells (Table 1). Then, a real-time quantitative reverse transcription (RT)-PCR assay was employed to validate the top three downregulated and top three upregulated genes (PDE1A, SLC7A7, ALDH2, SERPINB5, LUM, ING4) between the two cell lines. The expression of the 6 genes showed concordance with the microarray data ( Supplementary Figure S1B).

The Expression of ING4 Gene is Downregulated in SPC-A1/DTX and A549/Taxol Cells
Our gene expression data showed that ING4 was one of the top three    Figure 4B). Therefore, a decreasing level of ING4 expression oc-curred in lung adenocarcinoma cells in response to DTX.

Overexpression of ING4 Leads to the Increased Chemosensitivity of SPC-A1/DTX or A549/Taxol Cells to DTX
To investigate whether ING4 expression affects the sensitivity of lung adenocarcinoma cells to DTX, a pcDNA/ING4 vector expressing ING4 was stably transfected into SPC-A1/ DTX and A549/ Taxol cells. Compared with parental SPC-A1 or SPC-A1/DTX/ control cells, the level of ING4 mRNA or protein expression in SPC-A1/DTX/ ING4 cells was significantly increased (Figures 5A, B). Likewise, the level of ING4 mRNA or protein expression in A549/Taxol/ING4 cells was also significantly increased compared with A549 or A548/Taxol/ control cells (Figures 5A, B). Then, the IC 50 value of DTX or paclitaxel in SPC-A1/DTX or A549/Taxol cells was determined using an MTT assay. Compared with SPC-A1/ DTX/ control cells, the IC 50 value of DTX or paclitaxel in SPC-A1/ DTX/ING4 was significantly decreased by 58.6% or 36.8%, respectively ( Figure 5C). Compared with A549/Taxol cells, the IC 50 value of DTX or paclitaxel in A549/ Taxol/ ING4 cells was significantly decreased by 64.3% or 45.6%, respectively ( Figure 5C). Next, we analyzed cellcycle distribution in SPC-A1/ DTX/ ING4 or A549/Taxol/ING4 cells. Compared with the SPC-A1/DTX/control or A549/Taxol cell line, the SPC-A1/DTX/ ING4 or A549/ Taxol/ING4 cell line triggered an accumulation of cells at the G 2 /M stage, whereas the numbers of cells in S-phase and G 0 /G 1 phase accordingly decreased ( Figure 6A). Then, we examined the changes in apoptosis. Compared with SPC-A1/DTX/ control cells, the apoptotic rate of SPC-A1/ DTX/ ING4 cells was significantly enhanced by approximately 16.11% (p < 0.05; Figure 6B). These results strongly suggest that overexpression of ING4 could reverse the resistance of SPC-A1/ DTX cells to DTX or paclitaxel by induc- ing a G 2 /M-phase arrest and apoptosis enhancement.

Downregulation of ING4 Leads to the Decreased Chemosensitivity of SPC-A1 or A549 Cells to DTX
To extrapolate the finding that overexpression of ING4 could reverse the chemoresistance of SPC-A1/DTX or A549/Taxol cells, we employed RNA interference to downregulate ING4 expression and analyze the changes of chemo sensitivity in sensitive SPC-A1 or A549 cells. Compared with the SPC-A1/ shcontrol or A549/shcontrol cells, the level of ING4 mRNA and protein expression in SPC-A1/shING4 or A549/ shING4 cells was significantly decreased ( Figures 7A, B). Then, we determined the IC 50 value of DTX or paclitaxel using an MTT assay. Compared with SPC-A1/ shcontrol cells, the IC 50 value of DTX or paclitaxel in SPC-A1/shING4 cells was significantly increased by 39.6% or 73.7%, respectively (p < 0.05; Figure 7C). Additionally, compared with A549/ shcontrol cells, the IC 50 value of DTX or paclitaxel in A549/shING4 cells was significantly increased by 52.2% or 68.5%, respectively (p < 0.05; Figure 7C). Then, we analyzed the changes of cell-cycle distribution ( Figure 8A). Compared with SPC-A1/shcontrol or A549/shcontrol cells, the numbers of SPC-A1/shING4 or A549/shING4 cells in G 2 /M-phase accordingly decreased and the numbers of SPC-A1/shING4 or A549/shING4 cells in S-phase significantly increased (p < 0.05). However, the numbers of SPC-A1/ shING4 or A549/shING4 cells in G 0 /G 1 -phase showed no difference (p > 0.05). In addition, it was also shown that the numbers of DTX (0.08 μg/L)-treated SPC-A1 or A549 cells in G 2 /M-phase accordingly decreased and the numbers of DTX (0.08 μg/L)-treated SPC-A1 or A549 cells in S-phase significantly increased. In other previous research, it was shown that ING4 could impede the progression of the cell cycle by regulating the expression of cell cycle regulators (such as p27, cyclinD1 and SKP2), which led to cell arrest in G 2 /M phase (17). Next, flow cytometry was used to detect apoptosis. Compared with SPC-A1/shcontrol (or A549/shcontrol) cells, DTX-treated SPC-A1 (or A549) and SPC-A1/shING4 (or A549/shING4) cells showed no difference in apoptosis (p > 0.05; Figure 8B). Thus, the increased resistance of SPC-A1 or A549 cells to DTX or paclitaxel induced by ING4 downregulation was associated with a decreased number of cells in G 2 /M-phase and an increased number of cells in S-phase.

Effects of ING4 Expression on the Survival Pathway of SPC-A1/DTX Cells
To investigate further insight into the mechanism of apoptosis enhancement induced by overexpression of ING4, we first determined the involvement of caspase-3. Compared with SPC-A1/DTX/ control cells combined with DTX treatment (5.0 μg/L), the activation of caspase-3 protein was observed in SPC-A1/DTX/ING4 cells combined with DTX treatment (Figure 9A). Meanwhile, compared with SPC-A1/DTX/control cells combined with DTX treatment (5.0 μg/L), the activity of caspase-3 activity was significantly increased by approximately 251.3% in SPC-A1/DTX/ ING4 cells combined with DTX treatment (p < 0.01; Figure 9B). Then, we used a quantitative RT-PCR assay to detect the mRNA expression levels of some apoptosis-related genes, including survivin, Aurora-A, stathmin, XIAP, Bcl-2, Bcl-xL and Bax, which has been reported to be associated with the sensitivity of tumor  Figure 9C, SPC-A1/DTX/ING4 cells combined with DTX treatment showed a significant reduction in the level of Bcl-2 mRNA expression and a significant increase in the level of Bax mRNA expression ( Figure 9C). Western blot assay confirmed the decreased expression of Bcl-2 protein and the increased expression of Bax protein ( Figure 9D). Therefore, the decreased expression of Bcl-2 and increased expression of Bax in the SPC-A1/DTX/ING4 cells leads to the downregulated Bcl-2/Bax ratio, which finally induces the activation of caspase-3.

Overexpression of ING4 Increases In Vivo Chemosensitivity of SPC-A1/DTX Cells to DTX
The  Table S1; p < 0.05). Also, following treatment with DTX, TUNEL-staining assay showed that the rate of apoptotic tumor cells was significantly increased in tumors formed from SPC-A1/DTX/ING4 cells compared with that in tumors formed from SPC-A1/ DTX/ control cells ( Figure 10D). From these data, we concluded that overexpression of ING4 could increase the in vivo chemosensitivity of SPC-A1/DTX cells to DTX.

The Expression of ING4 Is Downregulated in DTX-Resistant Lung Adenocarcinoma Tissues
To investigate the association between ING4 expression in lung adenocarcinoma tissues and the clinical response to DTXbased regimens, semiquantitative immunohistochemistry on tumor biopsy specimens from 18 eligible patients with advanced lung adenocarcinoma treated with DTX combined with platinum agents was performed. As shown in Figure Fig ure 11C). Therefore, the expression of ING4 in advanced lung adenocarcinoma might be positively correlated with the response of patients to DTX.

DISCUSSION
Multiple factors associated with sensitivity of tumor cells to taxanes (paclitaxel and DTX) have been reported, including the MDR gene, alteration in tubulin dynamics, and differences in β-tubulin isotype expression (18,19). In particular, overexpression of β-tubulin III could induce paclitaxel or DTX resistance in association with reduced effects on microtubule dynamic instability, which suggests that upregulation of a neuronal tubulin isotype is a key contributor to taxane sensitivity in human cancers (20). Additionally, aberrant expression of some oncogenes could affect sensitivity of tumor cells to DTX (21)(22)(23). The Aurora-A gene is essential for the proper arrangement of centrosomes and microtubules, and overexpression of Aurora-A has been reported to induce increased resistance to taxanes via a decrease in spindle checkpoint activity in vitro (24). Other reports showed that suppression of Aurora-A expression could enhance chemosensitivity to DTX in human cancers, including esophageal squamous cell carcinoma, breast cancer and prostate cancer (25)(26)(27). Stathmin, also designated Op18, is a highly conserved ubiquitous cytoplasmic protein that has recently been shown to destabilize microtubules (28). Mutant p53 breast cancers exhibiting high levels of stathmin may be resistant to antimicrotubule agents and siRNA-mediated stathmin downregulation could enhance taxane chemosensitivity of tumor cells, including prostate cancer and osteosarcoma (29,30). Recently, epigenetic events, including DNA methylation and posttranscriptional regulation (microRNAs), were found to be involved in the control of gene expression, which is known to play an important role in cancer and chemotherapy drug resistance (31,32). Previously, we have identified microRNA expression profiles in DTX-resistant human NSCLC cells (SPC-A1), which provide a better understanding of mechanisms involved in drug sensitivity or resistance of NSCLC (33).
To further investigate the mechanisms of DTX resistance and provide theoretical support for drug-resistant reversal induced by DTX, a DTX-resistant variant of the human lung adenocarcinoma cell line SPC-A1 (SPC-A1/DTX) was previously established. The incipient concentration of DTX was 0.008 μg/L, and the SPC-A1/DTX cell line stably grew into 5.0 μg/L DTX. An MTT assay showed that the index of drug resistance of SPC-A1/DTX was 13.20 to DTX. The establishment of a DTX-resistant lung adenocarcinoma cell model provided the foundation for further research on its chemo resistant mechanisms. In the present study, we focused specifically on improving the sensitivity to DTX and the selection of a therapeutic program for each individual patient, to identify novel genes which define the cDNA expression profiles of the parental SPC-A1 cell line in comparison with its DTX-resistant subclone SPC-A1/DTX using a cDNA microarray. Then, we attempted to provide strong molecular, biochemical and biological evidence that downregulation of the ING4 gene played an important role in SPC-A1 cell progression to DTX resistance. ING4, a novel member of the ING family, which comprises six members characterized by a highly conserved C-terminal plant home domain (PHD)like zinc-finger domain, has been reported to be involved in a variety of processes including oncogenesis, angiogenesis and DNA repair (34 (36). And ING4 plus CDDP (cisplatin) was closely associated with the cooperative regulation of extrinsic and intrinsic apoptotic pathways and synergistic inhibition of tumor angiogenesis (37).
In the present study we aimed firstly to explore the roles of ING4 in DTX resistance of lung adenocarcinoma cells. We showed that the ING4 gene was significantly downregulated in DTXresistant SPC-A1 or taxol-resistant A549 cell lines when compared with nonresistant SPC-A1 or A549 cells at both transcriptional and translational levels. Ectopic expression of ING4 could significantly decrease the IC 50 value of SPC-A1/DTX or A549/Taxol cell lines, whereas siRNA-mediated ING4 downregulation could increase the IC 50 value of DTX-sensitive SPC-A1 or A549 cells. Overexpression of ING4 could inhibit in vivo growth of SPC-A1/DTX cells combined with DTX treatment. Moreover, we also found that a decreasing level of ING4 expression occurred in lung adenocarcinoma cells that responded to DTX. We then further elucidated the possible mechanisms of chemosensitivity enhancement induced by overexpression of ING4. Our results showed that overexpression of ING4 could significantly induce apoptosis enhancement and G 2 /M-phase arrest in SPCA-1/DTX cells, and the apoptosis enhancement might be associated with activation of caspase-3 induced by the decreased ratio of Bcl-2/ Bax. Next, we analyzed the effects of ING4 expression on the expression of some apoptosis-related genes such as survivin, XIAP, Bcl-xL, Bcl-2, Bax, Aurora-A and stathmin, which have been reported to affect the sensitivity of tumor cells to paclitaxel or DTX (38). We found that upregulation of ING4 could decrease Bcl-2 expression and increase Bax expression but had no effect on other genes at both transcriptional and translational levels. The decrease in the mitochondrial Bcl-2/Bax ratio is associated with caspase-3-dependent apoptosis, which is consistent with the findings of other researchers. Although Li and his colleagues showed that ING4 might play an inhibitory role in A549 cell prolifera-