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Therapeutic implications of enhanced G(0)/G(1) checkpoint control induced by coculture of prostate cancer cells with
1Johns Hopkins Oncology Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21231, USA.
Abstract:
Osteoblastic metastases are common in lethal prostate cancer. Effective therapy for bone metastases is lacking. Thus, developing an appropriate in vitro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming in vivo evaluation in bone metastases animal models. In the past, such in vitro screening evaluated the response of prostate cancer cells to chemotherapeutic agents in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e., >90%) proliferative growth fraction. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1%) in 117 metastatic sites of prostate cancer obtained from 11 androgen ablation failing patients at "warm" autopsy was found to be >10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human prostate cancer cells were cocultured with membrane-separated hFOB human osteoblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from >90 to <30%) without enhancing their low rate (i.e., <5%) of apoptosis. This lowering of the growth fraction was documented using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that coculture with osteoblasts causes enhanced p53, p27, and p21 expression leading to a decrease in the number of LNCaP cells entering the cell cycle (i.e., enhanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M and thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) checkpoint control affected the chemosensitivity of LNCaP cells. This was documented by coculturing LNCaP cells with hFOB cells to condition the medium for 3 days to lower the growth fraction to <30% before exposing the LNCaP cells for 48 h to various concentrations of Taxol, doxorubicin, or thapsigargin (TG). In standard high (i.e., >90%) growth fraction cultures (i.e., cultures in the absence of osteoblast-conditioned medium), there was a dose-dependent and significant (P < 0.05) increase in apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 microM) did not enhance apoptosis of lower growth fraction LNCaP cells cultured in osteoblast-conditioned medium. Similarly, only the highest concentration of doxorubicin (1 microM) enhanced apoptosis in lower growth fraction cells. In contrast, 100 nM TG induced high levels of apoptosis in both lower and high-growth fraction LNCaP cultures. These results demonstrate that the osteoblast/LNCaP coculture system is a better in vitro screen than monoculture to identify proliferation-independent agents for the treatment of prostate cancer bone metastases, and TG is such an agent.
Insights
Developing a better in vitro model for prostate cancer bone metastases is crucial. Coculturing prostate cancer cells with osteoblasts mimics clinical conditions, revealing that proliferation-independent agents like thapsigargin (TG) are more effective than traditional chemotherapy.
Area of Science:
- Oncology
- Cell Biology
- Cancer Metastasis
Background:
- Osteoblastic bone metastases are common in lethal prostate cancer, yet effective therapies are lacking.
- Current in vitro screening methods for prostate cancer agents do not accurately reflect the low proliferative fraction observed in clinical bone metastases.
- Accurate in vitro models are critical for prioritizing novel therapeutic agents for further in vivo evaluation.
Purpose of the Study:
- To develop a more clinically relevant in vitro model for screening prostate cancer bone metastases therapies.
- To investigate the effect of osteoblasts on prostate cancer cell proliferation and chemosensitivity.
- To identify therapeutic agents effective against low-proliferative prostate cancer cells in bone metastases.
Main Methods:
- Coculturing LNCaP human prostate cancer cells with hFOB human osteoblasts to mimic the bone microenvironment.
- Assessing the proliferative growth fraction of LNCaP cells using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation.
- Evaluating the chemosensitivity of LNCaP cells with varying growth fractions to Taxol, doxorubicin, and thapsigargin (TG) after exposure to osteoblast-conditioned medium.
Main Results:
- Coculturing LNCaP cells with osteoblasts significantly reduced their proliferative growth fraction (from >90% to <30%), mimicking clinical observations.
- Osteoblast coculture led to enhanced p53, p27, and p21 expression, increasing G(0)-G(1) cell cycle arrest.
- Traditional chemotherapeutic agents (Taxol, doxorubicin) showed reduced efficacy against low-proliferative cells, while thapsigargin (TG) effectively induced apoptosis in both high- and low-proliferative cells.
Conclusions:
- The osteoblast/LNCaP coculture system provides a superior in vitro screening platform for prostate cancer bone metastases compared to monoculture.
- This model highlights the importance of targeting proliferation-independent mechanisms for effective treatment of bone metastases.
- Thapsigargin (TG) demonstrates potential as a therapeutic agent for prostate cancer bone metastases due to its efficacy against low-proliferative cells.
