Our results show that IDA is cytotoxic to cell lines of murine Lewis Lung cancer and human prostate cancer DU-145 in vitro (Fig. ). The former arose spontaneously in a C57Bl/6 mouse as an anaplastic lung cancer. It was used for many years in cancer drug development by the National Cancer Institute, and was found to be the most refractory murine tumor in the screening program for effective chemotherapeutic agents [16
IDA is also cytotoxic to a variety of fresh human tumor samples in vitro (Table ). At the highest IDA concentration tested, 200 mcg/ml, only 1% of the cells of the five tumor specimens tested remained viable, but the deepest dose responses occurred in untreated gastric cancer and previously treated breast and prostate cancer specimens. Previously treated non-Hodgkin’s lymphoma, and an ovarian cancer as well as an untreated poorly differentiated tumor of unknown primary site were the most sensitive tumors. Whereas a previously treated ovarian tumor as well as previously treated colon cancer and adenocarcinoma of unknown primary site specimens were relatively resistant.
The ability to predict in vivo responses of patients from in vitro chemosensitivity testing of their tumors has been intensively studied. The DiSC assay is one of a class of fresh tumor, primary culture assays measuring cell death as an endpoint. Other assays of this class include the MTT assay, ATP assay, and fluorescein diacetate assay. The above four assays have been compared with each other and have been found to give similar results in fresh tumor specimens in cases where more than 70% of the viable cells present are tumor cells [18
]. The major advantage of the DiSC assay is that it provides the cell death endpoint which is most specific for drug effects on tumor cells, in a mixed population of tumor and normal cells. Cell death endpoints have been consistently found to predict for both patient response to chemotherapy and for patient survival with chemotherapy, in 40 published studies in more than 2,000 patients [19
]. Therefore, the DiSC assay results reported in Table suggest that IDA will prove to be an effective clinical chemotherapeutic agent.
There is a considerable clinical experience with the use of IDA. During the 1980s and 1990s IDA was studied in patients with a variety of neuropsychiatric disorders, including Alzheimer’s dementia, bipolar depression, schizophrenia, supranuclear palsy, and Parkinson’s disease [20
]. IDA dosing could be tolerated at 240 mg daily with minimal side effects of nausea and asymptomatic decline in standing diastolic blood pressure [21
]. No pancreatic toxicities were described in the use of IDA in neuropsychiatric patients. However, since the maximum tolerated dose of IDA has not been defined, a Phase I trial would be required before specific trials of drug efficacy could be carried out in patients with malignancies.
Many effective chemotherapeutic drugs produce tumor apoptosis [25
]. Flow cytometric analysis using annexin V expression shows that 96% of 3LL cells become apoptotic after 24-h exposure to IDA. Recent studies have shown that some, but not all, apoptotic promoting chemotherapeutic drugs enhance membrane expression of calreticulin, the cytoplasmic chaperone protein. Since it is appreciated that calreticulin expressing apoptotic tumor cells are frequently immunogenic and are recognized by the host immune system leading to tumor rejection [26
], we defined calreticulin expression by 3LL and DU-145 cells after exposure to IDA. Ninety percent of 3LL cells and 40% of DU-145 cells overexpress calreticulin after 24-h incubation with IDA from baselines of less than 3 and 9%, respectively.
Since chemotherapeutic agents kill tumors by first-order kinetics, it has been suggested that tumor cure depends on the host immune response to eradicate residual viable tumor [28
]. Recent observation regarding chemotherapeutically induced calreticulin overexpression by apoptotic tumor cells support this concept [26
]. The ability of IDA to produce calreticulin overexpression in 3LL and DU-145 suggest that it might promote immune recognition and rejection of syngeneic tumors by an intact host.
Etoposide is an immunosuppressive topoisomerase II inhibiting drug that is widely used clinically to treat a variety of tumors, including leukemia, lymphoma, lung, and testicular cancer [31
]. It impairs the integrity of DNA strand replication, promotes apoptosis and is used as a positive control for calreticulin expression induction [25
]. We have previously shown that etoposide produces important immune activating effects in vivo leading to rejection of murine models of leukemia and lung cancer [33
]. Mice bearing L1210 acute lymphatic leukemia which had achieved a sustained response to etoposide rejected a subsequent L1210 challenge [33
]. This effect is tumor specific and mediated by CD8-T lymphocytes [35
]. We have also shown that mice bearing 3LL effectively treated with etoposide behave similarly. In addition, the majority of naïve mice that had survived an inoculation of viable 3LL cell, which had previously been incubated in vitro with etoposide rejected challenge with wild type 3LL [34
Although the biochemical mechanism by which IDA promotes tumor cell death is not known, our work shows that IDA has antineoplastic activity in vitro and its cytotoxic effect is produced apoptotically. This effect is associated with a marked induction of calreticulin membrane expression, suggesting that in addition to its potential development as a cytotoxic antineoplastic agent, IDA may also prove useful in initiating host immunologic responses against autologous tumors. In vivo studies exploring these possibilities are underway. Our preliminary results in this area are encouraging since we have observed a 50% reduction in the growth of 3LL tumor in mice treated with IDA, and delay in the growth of wild type 3LL cells in syngeneic host mice previously inoculated with calreticulin expressing 3LL cells conditioned in vitro with IDA.