Wolfram Dempke

Epidemiologic studies have documented a 40–50% reduction in incidence of colorectal cancer in individuals taking nonsteroidal antiinflammatory drugs (NSAIDs). Since NSAIDs are known to inhibit cyclooxygenases (COX-1, COX-2), the basic mechanism of their antitumor effects is conceivably the altered metabolism of arachidonic acid and, subsequently, prostaglandins (PGs). Although COX-2, the inducible isoform, is regularly expressed at low levels in colonic mucosa, its activity increases dramatically following mutation of the APC (adenomatous polyposis coli) gene suggesting that β-catenin/T-cell factor mediated Wnt-signaling activity may regulate COX-2 gene expression. In addition, hypoxic conditions and sodium butyrate exposure may also contribute to COX-2 gene transcription in human cancers. The development of selective COX-2 inhibitors has made it possible to further evaluate the role of COX-2 activity in colorectal carcinogenesis. To date, at least five mechanisms by which COX-2 contributes to tumorigenesis and the malignant phenotype of tumor cells have been identified, including: (1) inhibition of apoptosis; (2) increased angiogenesis; (3) increased invasiveness; (4) modulation of inflammation/immuno-suppression; and (5) conversion of procarcinogens to carcinogens. A clear positive correlation between COX-2 expression and inhibition of apoptosis has been established, associated with increased PGE₂ levels resulting in modulation of pro- and anti-apoptotic factors (e.g., bcl-2, MAKs/ras, caspase-3, Par-4). In terms of angiogenesis and invasiveness, COX-2 activity was found to increase the expression of growth factors (e.g., VDEG, PDGF, bFGF) and matrix metalloproteinases (MMPs). Since COX-2 inhibitors have been demonstrated to interfere with tumorigenesis and apoptosis, COX-2 and its gene product may be attractive targets for therapeutic and chemoprotective strategies in colorectal cancer patients. This may lead to new perspectives that by controlling the cancer phenotype, rather than attempting to eradicate all affected cells, may provide significant benefits to the cancer patient.

Human germ cell tumors have the unique capacity for totipotential differentiation. AFP (the product of normal yolk sac) and HCG (produced by trophoblastic tissues) are frequently produced by germ cell tumors. The a-subunit of the glycoprotein HCG is identical to that of several pituitary glycoprotein hormones (e.g. TSH, LH, FSH), whereas the b-subunit of HCG, TSH, LH and FSH is homologous but distinct in the terminal amino acid sequence suggesting that HCG is part of a superfamily of gestational hormones. However, the role of TSH within this hormone superfamily is still not yet established. A 24-year old patient was admitted to our clinic because of a widespread recurrence of a germ cell tumor (stage IIIC, Lugano classification). The routine hematologic and blood chemical tests were normal, yet, an elevated HCG was found. In addition, increased levels of the thyroid hormones FT3 and FT4 were seen, although, this was not associated with clinical symptoms of a hyperthyreosis. There was no history of hyperthyreosis and thyroidal autoantibody screening revealed normal titers. An ultrasound examination of the thyroid gland showed no abnormalities and no iodine exposure had occurred during the last months. To mobilize peripheral stem cells (PBSC) he was initially treated with paclitaxel (175 mg/m2) and ifosfamide (8.000 mg/m2)) followed by apheresis of PBSC. The patient was then entered in our phase-II-study for relapsing germ cell carcinomas using a high-dose chemotherapy regime (paclitaxel 175 mg/m2, ifosfamide 9.000 mg/m2, carboplatin 900 mg/m2, etoposide 900 mg/m2) with subsequent retransfusion of collected stem cells. Due to cranial metastases an cranial irradiation was also performed. After three courses of this protocol an excellent partial remission of the tumor lesions was achieved and the HCG value dramatically decreased. Due to elevated thyroidal hormones, the patient was initially treated with thiamazole (20 mg) resulting in decrease of the thyroidal hormones. Thus, the thiamazole dose was reduced to 5 mg and then omitted. The decrease of the thyroidal hormones FT3 and FT4 strongly correlated with the reduction of HCG values (r2 0.91 and 0.77, p < 0.0008). To date there is only slight evidence that enhanced HCG levels may cause, at least in part, a hyperthyreosis (e.g. gestational hyperthyreosis), however, the underlying biochemical mechanism still remains unclear. In this case report we have demonstrated a clear positive correlation between HCG levels and thyroidal hormones in a patient with germ cell tumor suggesting a direct stimulation of hormone producing thyroidal cells by HCG, however, this was not associated with clinical symptoms of hyperthyreosis. Currently, several in vitro studies are underway in our laboratory to further elucidate the biochemical mechanisms of HCG induced hyperthyreosis.

Microangiopathic hemolytic anemia (MAHA) and disseminated intravasal coagulation (DIC) as initial paraneoplastic symptoms of a solid tumor present a rare clinical situation. In 1998 a female patient was admitted due to multiple thrombosis, thrombocytopenia and fever. The initial diagnostic procedures revealed peri-aortic lymphomas and a tumor bulk (7 x 8 cm) in the upper abdomen. Gastroscopy revealed a 2 cm ulcer at the back side of the gastric corpus. Histologically, a signet-ring cell carcinoma was diagnosed. Final diagnosis stated a multilocular metastasising gastric cancer with infiltration of bone, peritoneum and dura and signet-cell infiltration of the bone marrow. Hematologic investigation in view of multiple paraneoplastic thrombosis revealed a microangiopathic hemolytic anemia associated with disseminated intravasal coagulation. Parallel to initial symptomatic therapy of coagulopathy, systemic cytostatic therapy with CDDP and VP-16 was initiated. In addition, radiotherapy of the brain was performed. After histologic confirmation of the diagnosis, weekly therapy with 5-FU (2600 mg/m2) and folinic acid (500 mg/m2) according to the Ardalan protocol was performed. After first signs of moderate response, oxaliplatin (60 mg/m2, day 1) was added. Although the chemotherapy dose had to be reduced due to prolonged neutropenia, the disturbances of hemostasis resolved completely resulting in reduced substitution rates with fresh frozen plasma (FFP) and platelets. Unfortunately, the patient died at home due to pulmonary embolism. Tumor-associated hemostaseologic alteration requires immediate substitution of FFP and platelets. However, it should be followed by specific therapy of malignancy, since tumor-induced metabolites (e.g. mucin) maintain the alteration of hemostasis. Chemotherapy may therefore be the best strategy to prevent complications such as MAHA and DIC.

Cisplatin is among the most widely used broadly active cytotoxic anticancer drugs; however, its clinical efficacy is often limited by primary or the development of secondary resistance. Several mechanisms have been implicated in cisplatin resistance, including reduced drug uptake, increased cellular thiol/folate levels and increased DNA repair. More recently, additional pathways have been characterized indicating that altered expression of oncogenes that subsequently limit the formation of cisplatin-DNA adducts and activate anti-apoptotic pathways may also contribute to the resistance phenotype. Several lines of evidence suggest that expression of ras oncogenes can confer resistance to cisplatin by reducing drug uptake and increasing DNA repair; however, this is not a uniform finding. Tumor cells, in contrast to normal cells, respond to cisplatin exposure with transient gene expression to protect or repair their chromosomes. The c- fos /AP-1 complex, a master switch for turning on other genes in response to DNA-damaging agents, has been shown to play a major role in cisplatin resistance. In addition, AP-2 transcription factors, modulated by protein kinase A, are also implicated in cisplatin resistance by regulating genes encoding for DNA polymerase β and metallothionines. Furthermore, considerable evidence indicates that mutated p53 plays a significant role in the development of cisplatin resistance since several genes implicated in drug resistance and apoptosis (e.g. mismatch repair, bcl -2, high mobility group proteins, DNA polymerases α and β, PCNA, and insulin-like growth factor) are known to be regulated by the p53 oncoprotein. Improved understanding of molecular factors for the development of cisplatin resistance may allow the prediction of clinical response to cisplatin-based treatment. Furthermore, the identification of oncogenes involved in cisplatin resistance has already led to in vitro approaches which successfully inactivated these genes using ribozymes or antisense oligodeoxynucleotides, thus restoring cisplatin sensitivity. It is conceivable that these strategies, once transferred to a clinical setting, may have the potential to enhance the efficacy of cisplatin against a great variety of malignancies and thus more fully exploit the antineoplastic and curative potential of this drug.

For nearly four decades, 5-fluorouracil (5-FU) has been the mainstay of treatment for colorectal cancer. Due to the lack of other agents with significant activity, tremendous efforts have been undertaken to increase the efficacy of 5-FU by investigating alternative schedules of delivery and biomodulation. However, bolus 5-FU in combination with folinic acid (FA), either as the Mayo Clinic or Roswell Park protocol, still represents the standard treatment for adjuvant and first-line palliative chemotherapy of colorectal cancer. In a recent meta-analysis, infusional protocols of 5-FU demonstrated increased response rates (14% to 22%) and a marginal, but significant survival benefit of 3 weeks (11.3 to 12.1 months). In view of the much higher costs and complicated management of infusional 5-FU regimens, this marginal survival benefit does not yet allow protracted 5-FU application to be defined as standard therapy. However, protracted 5-FU infusion in combination with radiation can be considered standard therapy as adjuvant treatment of rectal cancer, since it has demonstrated a significant increase in survival. In the future, oral 5-FU prodrugs may be substituted for infusional 5-FU. Furthermore, current data indicate that 5-FU will also be an essential component of combination chemotherapy protocols with the new active agents oxaliplatin, irinotecan, and raltitrexed. Preclinical studies show synergistic antitumor activity of 5-FU with these agents, which corresponds well with clinical response rates of 50% in untreated and 15% to 25% in 5-FU-refractory patients. Moreover, 5-FU-based pro-drug-active drug systems serve as excellent models for tumor-targeted gene therapy.

In vitro exposure of the TR170 ovarian carcinoma cell line to six intermittent 24-h treatments with a 90% inhibitory concentration of cisplatin (CDDP) (0.15 micrograms/ml; 0.5 microM) resulted in a 2-fold stably resistant subline designated TR170/CP+ (B.T. Hill et al., Int. J. Cancer, 39: 219-225, 1987). Resistance to CDDP in these CP+ cells has now been associated with reduced uptake of 195mCDDP (2-fold; P less than 0.01) and decreased removal of specific Pt-DNA adducts, quantitated immunochemically, indicative of an apparent increased tolerance of CDDP-induced DNA damage. Specifically these resistant cells appeared deficient in removal of the major cis-Pt-(NH3)2d(pGpG) adduct and the difunctional cis-Pt(NH3)2d(GMP)2 lesion, showed less efficiency in removing cis-Pt(NH3)2d(pApG) adducts, but proved as proficient as the parental cell line in removing DNA-DNA interstrand cross-links. Activities of DNA polymerase-alpha and -beta were comparable in both lines, and no significant alterations in glutathione metabolism were identified. Response to acute X-irradiation was not modified in these TR170/CP+ cells, but they showed marked (10-fold) cross-resistance to 5-fluorouracil and, unusually, proved collaterally sensitive (12-fold) to methotrexate. Resistance to 5-fluorouracil was associated with significantly increased thymidylate synthase activity (P less than 0.01), but this was not reflected in altered gene expression, while increased sensitivity to methotrexate was accompanied by increased drug uptake but by unaltered activity and expression of dihydrofolate reductase. These results indicate that exposure to CDDP can result in numerous alterations, both intracellularly and at the cellular membrane, reflected in significant changes in the tumor cells' responses to the cytotoxic effects of a range of antitumor drugs. The clinical relevance of these observations remains to be established.

Examples of collateral sensitivity, even in experimental tumor systems, remain few. Preliminary data from this laboratory indicated that certain tumor cells expressed increased sensitivity to cisplatin after exposure in vitro to x-irradiation. To further clarify whether the type of fractionated radiation procedure used clinically can induce hypersensitivities to certain antitumor drugs we have pre-exposed the human ovarian carcinoma cell line JA-T/P derived from a tumor from an untreated patient to fractionated x-irradiation (total dose 50 Gy) in vitro. The resultant subline JA-T/DXR-10 expressed collateral sensitivity to cisplatin (CDDP), methotrexate (MTX) and fluorouracil (5-FU), but not to acute x-irradiation. Hypersensitivity to CDDP was associated with decreased activity of DNA polymerase beta (3.5-fold, P less than .01), but unaltered glutathione metabolism. Pre-incubation with cyclosporin A or with 3-aminobenzamide significantly enhanced (twofold, P less than .01) CDDP-induced cytotoxicity in JA-T/P cells, but not in the DXR-10 subline. Consistent with MTX hypersensitivity dihydrofolate reductase activity was significantly decreased (2.9-fold, P less than .01). Despite collateral sensitivity to 5-FU, however, thymidylate synthase activity was increased (twofold, P less than .05) suggesting alternative mechanisms for 5-FU-induced cytotoxicity in these JA-T/DXR-10 cells. These data demonstrate that DNA repair and associated reduced folate metabolism can be modified not only by drugs but also by fractionated x-irradiation.