Elsevier

Lung Cancer

Volume 59, Issue 2, February 2008, Pages 180-191
Lung Cancer

Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival for non-small-cell lung cancers

https://doi.org/10.1016/j.lungcan.2007.08.026Get rights and content

Summary

Background

Although zoledronic acid (ZOL), a third-generation nitrogen-containing bisphosphonate, has been identified as an attractive therapeutic agent against breast cancer, prostate cancer, multiple myeloma as well as small-cell lung cancer (SCLC), as best as we are aware, the anti-tumor effect of ZOL upon non-small-cell lung cancer (NSCLC) remains to be effectively investigated. This study examined the effects of ZOL upon the line-1 tumor cell, using a murine lung adenocarcinoma cell line similar to the behavior of human lung adenocarcinoma.

Methods

We investigated the anti-tumor effects of ZOL (3–100 μM) on line-1 tumor cells in vitro, including cellular proliferation, by means of an MTT assay, cell-cycle analysis by flow cytometry and by assessing the level of apoptosis by annexin V/propidium iodide (PI) and 4′-6-diamidino-2-phenylindole (DAPI) staining. Further, we evaluated the growth and survival of line-1 tumor cells following ZOL treatment (1 μg/kg/week) using an animal model. We also examined the in vivo cell-cycle pattern using lacZ-expressing line-1 cells (line-1/lacZ).

Results

ZOL significantly slowed the line-1 tumor growth in a dose-dependent manner in vitro. The treated line-1 tumor cells typically arrested at the S/G2/M-phase of the cell-cycle following ZOL exposure, but no apoptotic cells could be detected by either annexin V/PI or DAPI staining. When the ZOL was washed out, the drug-inhibited cells continued to proliferate again and the cell-cycle prolongation elicited earlier by the drug, then disappeared. Within 72–96 h following drug removal, the cell-cycle of the treated cells revealed a similar distribution to that of the untreated controls. Invivo studies demonstrated that ZOL significantly slowed the line-1 tumor growth. Indeed, mice lived significantly longer when they had been ZOL-treated than was the case for untreated mice (p < 0.05). Using line-1/lacZ cells, the in vivo cell-cycle distribution of line-1 tumor cells subsequent to ZOL exposure revealed S/G2/M-phase arrest that was identical to the in vitro culture.

Conclusions

ZOL maintains the potential to reduce tumor burden and prolong survival for murine pulmonary adenocarcinoma. The flow cytometrical analysis of cell-cycle demonstrated that ZOL induces no apoptosis but is able to arrest line-1 tumor cells at the S/G2/M-phase. Although the clinical relevance of these results warrants verification for human lung cancer patients, ZOL combined with chemotherapy and/or radiotherapy appears to be a new therapeutic strategy for the effective treatment of NSCLC.

Introduction

Zoledronic acid (ZOL), a third-generation of nitrogen-containing bisphosphonate, has been used clinically to treat malignancy-induced bone and metabolic disorders such as bone pain, pathological bone fracture and hypercalcemia, by way of its modulation of osetoclast activity [1], [2], [3]. Recently, an increasing number of preclinical studies have shown that ZOL exerts anti-tumor activity against a variety of malignancies including breast cancer, prostate cancer, pancreatic cancer, melanoma, osteosarcoma, multiple myeloma and chronic myeloid leukemia [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. ZOL induces apoptosis, inhibits cellular proliferation, prevents angiogenesis and impedes tumor cell invasion to the extraceullar matrix, thus it slows tumor growth and decreases metastases [4], [5], [6], [9], [14], [15], [16], [17]. The inhibitory effect of ZOL upon the proliferation of certain cancer cells has been reported to be enhanced when combined with the use of anti-neoplastic agents, such as gemcitabicine, irinotecan, taxanes and anthracyclines [14], [18], [19], [20], [21].

Non-small-cell lung cancer (NSCLC) has been placed on the top of the list of cancer incidence and cancer mortality in a number of different countries for decades, this disease being responsible for more deaths than breast cancer, colon cancer or prostate cancer. Although surgery offers a reasonable curative rate for early-stage lung cancer patients, the overall 5-year survival for advanced lung cancer patients including unresectable stage-III and stage-IV tumors is less than 5% [22]. Intensive chemotherapy and radiotherapy have, in the past, been attempted with this malady in order to try to prolong the afflicted-individual's life span, but the outcome of such a therapeutic regimen has generally been unsatisfactory; these patients have often been dying of treatment-related complications and/or para-neoplastic syndrome. It has been previously estimated that approximately 30–65% of NSCLC cancer patients present with metastatic bone events at the time of diagnosis, such a secondary outcome ranking in frequency only behind that for liver and lung primaries [23], [24]. Because the dual activity of ZOL includes anti-osteoclast and anti-tumor activity, ZOL has been a not-unreasonable therapeutic candidate for NSCLC. On the other hand, ZOL has demonstrated inhibition of small-cell lung cancer (SCLC) tumor growth in an animal model and has also been reported to elicit a reduction in cancer-induced skeletal complications during a number of human lung cancer clinical trials [20], [25].

From a review of the relevant literature, it would appear that some intriguing phenomena concerning the anti-tumor activity of ZOL against examples of lung cancer have not been adequately addressed [20], [26]. Firstly, compared with studies of breast and prostate cancers the authors of which have reported that apoptosis is significantly associated with anti-proliferation of tumor cells, the proapoptotic activity of ZOL appears to be less correlated in lung cancer. Secondly, to the best of our knowledge, all studies regarding the anti-tumor mechanisms of ZOL were based upon a limited field of in vitro experiments. As best as we are aware, there exists no literature-based example of studies investigating the corresponding in vivo mechanism for an animal model. Direct extrapolation of in vitro data derived from cell culture experiments to an in vivo condition may not be appropriate. Additionally, previous investigation has revealed that ZOL administration may elicit the efficient prevention of skeletal complications but appeared to be unable to improve patient survival by human clinical trial [25]. Again, to the best of our understanding, there would appear to be an absence of animal-experiment studies reported in the literature that have noted an extended survival for lung cancer patients following ZOL treatment. Finally, as best we are aware, at present, it remains uncertain as to whether continuous exposure to ZOL is required for maintaining its anti-tumor activity against lung cancer.

In the present study, we have attempted to evaluate the effect of ZOL upon NSCLC. Herein, we utilized the line-1 tumor cell line, a murine lung cancer cell line, to investigate the outcome of apoptotic conditions, cell-cycle activity and the survival of lung cancer sufferers following ZOL treatment. The previous studies have shown that the line-1 tumor cell line was utilized to study tumor behavior, specially tumor metastasis and immune response in mouse model [27], [28], [29], [30], [31]. The tumor behavior of line-1 cells is, reportedly, similar to that of human pulmonary adenocarcinoma [30]. This cell line is highly tumorigenic in that less than 100 tumor cells are required to form progressive tumors growing in 50% of animals, and the tumor naturally metastasizes to the liver and lung [30]. The aggressive nature of the line-1 tumor is such that it may be appropriately used to simulate the clinical status of human NSCLC for experimental studies. Using the line-1 cell system, we found that ZOL inhibited existing lung cancer growth via cell-cycle blockage at the S/G2/M-phase both in vitro as well as in vivo. The tumor-bearing mice participating in this study indeed lived longer following ZOL treatment than was the case for the controls, however, the tumor-inhibitory effect of ZOL would appear to be short-lived if the drug is not continued.

Section snippets

Animal

BALB/cByJ mice, 6–8 weeks of age, were purchased from the Taiwan National Laboratory Animal Center. Guidelines for humane treatment of animals were followed, as approved by the Committee for Animal Resources at the Chang Gung Memorial Hospital and the Chang Gung University.

Cell line

Line-1 cancer cells, derived from BABB/cByJ mice, were originally obtained from Dr. John Yuhas [30]. This cell line was maintained in complete medium using RPMI 1640 culture medium (Bio West, France) supplemented with 10%

Zoledronic acid induces an anti-proliferative effect upon line-1 tumor cells in vitro

We utilized a MTT assay to investigate whether ZOL affected the proliferation of line-1 cancer cells in vitro. Line-1 cells were treated with increasing concentrations of ZOL for either 48 or 72 h. As revealed in Fig. 1, line-1 tumor cell growth was inhibited by ZOL in a concentration-dependent manner. The tumor cell growth was reduced to 60% of the control value at a ZOL concentration of 50 μM and further inhibited to 85% of the control value at a ZOL concentration of 100 μM over a period of

Discussion

In the present study, we clearly demonstrated that ZOL elicited an anti-proliferative effect upon tumor cell growth, inhibited tumor growth in general, and extended survival of mice afflicted with lung adenocarcinoma. Further, the tumor-inhibitory effect driven by ZOL would appear to be reversible once the drug is discontinued. We have also shown that ZOL is unable to induce apoptosis but the cell-cycle prolongation in line-1 tumor cells. Using line-1/lacZ cells, the present work provided the

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

The study was supported by a grant (NMRPG330062) from the National Science Council, Taiwan, and a grant (CMRPG350381) from the Chang Gung Memorial Hospital.

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