Viney Jain

IMPROVING CANCER RADIOTHERAPY WITH 2-DEOXY-D-GLUCOSE: PHASE I/II CLINICAL TRIALS ON HUMAN CEREBRAL GLIOMAS

PURPOSE Evaluation of tolerance, toxicity, and feasibility of combining large fraction (5 Gy) radiotherapy with 2-deoxy-D-glucose (2DG), an inhibitor of glucose transport and glycolysis, which has been shown to differentially inhibit repair of radiation damage in cancer cells. METHODS AND MATERIALS Twenty patients with supratentorial glioma (Grade 3/4), following surgery were treated with four weekly fractions of oral 2DG (200 mg/kg body weight) followed by whole brain irradiation (5 Gy). Two weeks later, supplement focal radiation to the tumor (14 Gy/7 fractions) was given. Routine clinical evaluation, x-ray computerized tomography (CT), and magnetic resonance (MR) imaging were carried out to study the acute and late radiation effects. RESULTS All the 20 patients completed the treatment without any interruption. The vital parameters were within normal limits during the treatment. None reported headache during the treatment. Mild to moderate nausea and vomiting were observed during the days of combined therapy (2DG + RT) in 10 patients. No significant deterioration of the neurological status was observed during the treatment period. Seven patients were alive at 63, 43, 36, 28, 27, 19, and 18 months of follow-up. In these patients, the clinical and MR imaging studies did not reveal any late radiation effects. CONCLUSIONS Feasibility of administering the treatment (2DG + 5 Gy) is demonstrated by the excellent tolerance observed in all 20 patients. Further, the clinical and MR studies also show the absence of any brain parenchymal damage.

11C-l-Methionine Positron Emission Tomography in the Clinical Management of Cerebral Gliomas

Positron emission tomography (PET) using l-[methyl-11C]-methionine (MET) is the most popular amino acid imaging modality in oncology, although its use is restricted to PET centers with an in-house cyclotron facility. This review focuses on the role of MET–PET in imaging of cerebral gliomas. The biological background of tumor imaging with methionine is discussed with particular emphasis on cellular amino acid transport, amino acid utilization in brain, normal metabolism of methionine, and its alterations in cancer. The role of MET–PET in clinical management of cerebral gliomas in initial diagnosis, differentiation of tumor recurrence from radiation injury, grading, prognostication, tumor-extent delineation, biopsy planning, surgical resection and radiotherapy planning, and assessment of response to therapy is also reviewed in detail.

Optimizing Cancer Radiotherapy with 2-Deoxy-D-Glucose

Background and Purpose:Higher rates of glucose utilization and glycolysis generally correlate with poor prognosis in several types of malignant tumors. Own earlier studies on model systems demonstrated that the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) could enhance the efficacy of radiotherapy in a dose-dependent manner by selectively sensitizing cancer cells while protecting normal cells. Phase I/II clinical trials indicated that the combination of 2-DG, at an oral dose of 200 mg/kg body weight (BW), with large fractions of γ-radiation was well tolerated in cerebral glioma patients. Since higher 2-DG doses are expected to improve the therapeutic gain, present studies were undertaken to examine the tolerance and safety of escalating 2-DG dose during combined treatment (2-DG + radiotherapy) in glioblastoma multiforme patients.Patients and Methods:Untreated patients with histologically proven glioblastoma multiforme (WHO criteria) were included in the study. Seven weekly fractions of 60Co γ-rays (5 Gy/fraction) were delivered to the tumor volume (presurgical CT/MRI evaluation) plus 3 cm margin. Escalating 2-DG doses (200–250–300 mg/kg BW) were administered orally 30 min before irradiation after overnight fasting. Acute toxicity and tolerance were studied by monitoring the vital parameters and side effects. Late radiation damage and treatment responses were studied radiologically and clinically in surviving patients.Results:Transient side effects similar to hypoglycemia were observed in most of the patients. Tolerance and patient compliance to the combined treatment were very good up to a 2-DG dose of 250 mg/kg BW. However, at the higher dose of 300 mg/kg BW, two out of six patients were very restless and could not complete treatment, though significant changes in the vital parameters were not observed even at this dose. No significant damage to the normal brain tissue was observed during follow-up in seven out of ten patients who received complete treatment and survived between 11 and 46 months after treatment.Conclusion:Oral administration of 2-DG combined with large fractions of radiation (5 Gy/fraction/week) is safe and could be tolerated in glioblastoma patients without any acute toxicity and late radiation damage to the normal brain. Further clinical studies to evaluate the efficacy of the combined treatment are warranted.Hintergrund und Ziel:Erhöhter Glukoseverbrauch und verstärkte Glykolyse korrelieren bei einigen Malignomformen generell mit schlechter Prognose. Eigene Untersuchungen an Modellsystemen zeigten, dass die nichtmetabolisierbare Glukoseverbindung 2-Deoxy-D-Glukose (2-DG) die Wirksamkeit der Strahlentherapie dosisabhängig verstärken kann, indem sie selektiv Krebszellen sensibilisiert, dagegen auf normale Zellen protektiv wirkt. Klinische Phase-I/II-Studien sprechen dafür, dass die Kombination von 2-DG (in einer oralen Dosis von 200 mg/kg Körpergewicht) mit großen Fraktionen einer γ-Strahlung von Patienten mit Hirntumoren gut vertragen wird. Da man erwartet, dass höhere 2-DG-Dosierungen das therapeutische Ansprechen verbessern, wurden die hier vorgestellten Untersuchungen unternommen, um an Glioblastoma-multiforme-Patienten Verträglichkeit und Sicherheit der Dosiseskalation von 2-DG in der Kombinationsbehandlung (2-DG + Radiotherapie) zu prüfen.Patienten und Methodik:In die Studie wurden nicht vorbehandelte Patienten mit histologisch belegtem Glioblastoma multiforme (WHO-Kriterien) eingeschlossen. Das Tumorvolumen (gemäß präoperativer CT/MRT-Auswertung) plus ein 3-cm-Sicherheitssaum wurden wöchentlich mit sieben Fraktionen einer 60Co-γ-Strahlung (5 Gy/Fraktion) behandelt. In ansteigender Dosierung wurde 2-DG (200–250–300 mg/kg KW) nach nächtlicher Nahrungskarenz 30 min vor der Bestrahlung verabreicht. Akuttoxizität und Verträglichkeit wurden durch Überwachung von Vitalparametern und Nebenwirkungen untersucht. Langzeit-Strahlenfolgen und Ansprechen wurden bei überlebenden Patienten radiologisch und klinisch untersucht.Ergebnisse:Bei den meisten Patienten wurden vorübergehende, Hypoglykämie-ähnliche Symptome beobachtet. Verträglichkeit und Patienten-Compliance der Kombinationsbehandlung waren bis zu einer 2-DG-Dosis von 250 mg/kg KG sehr gut; bei der höheren Dosis von 300 mg/kg KG entwickelten zwei von sechs Patienten jedoch starke Unruhe und konnten die Behandlung nicht abschließen, obwohl auch unter dieser Dosis keine signifikanten Veränderungen der Vitalparameter festgestellt wurden. Im Follow-up war keine signifikante Schädigung des normalen Hirngewebes bei sieben von zehn Patienten zu beobachten, die die komplette Behandlung erhalten und zwischen 11 und 46 Monate nach der Behandlung überlebt hatten.Schlussfolgerung:Die orale Gabe von 2-DG kombiniert mit hohen Einzelfraktionen (5 Gy/Fraktion/Woche) ist sicher und wurde von Gliom-Patienten ohne Akuttoxizität und ohne Strahlungsspätschäden im Hirngewebe vertragen. Um die Wirksamkeit der Kombinationstherapie zu prüfen, sind weitere klinische Studien erforderlich.

Clinical studies for improving radiotherapy with 2-deoxy-D-glucose: Present status and future prospects

Higher rates of glucose usage generally correlate with poor prognosis in several types of malignant tumours. Experimental studies (both in vitro and in vivo) have shown that 2-deoxy-D-glucose (2-DG), a glucose analog and glycolytic inhibitor, enhances radiation-induced damage selectively in tumor cells while protecting normal cells, thereby suggesting that 2-DG can be used as a differential radiomodifier to improve the efficacy of radiotherapy. Clinical trials undertaken to study the feasibility, safety, and validity of this suggested approach will be described. Based on 2-DG-induced radiosensitization observed in primary organ cultures of cerebral glioma tissues, clinical trials were designed taking into consideration the radiobiology of gliomas and pharmacokinetics of 2-DG. Phase I/II clinical trials have unequivocally demonstrated that a combination of 2-DG (200-300 mg 2-DG per kg body weight orally administered after overnight fasting, 20 min before irradiation) with large weekly fractions (5 Gy/fraction) of low-LET radiotherapy is well tolerated without any acute toxicity or late radiation damage to the normal brain tissue. Nonserious transient side effects similar to hypoglycemia induced disturbances like restlessness, nausea, and vomiting were observed at the 2-DG doses used. Data from these trials involving more than 100 patients have clearly indicated a moderate increase in the survival, with a significant improvement in the quality of life with clinicopathological evidence of protection of normal brain tissue. A phase III multicentric trial to evaluate the efficacy of the combined treatment is in progress. Directions for future studies are discussed.

Low-dose radiation hypersensitivity in human tumor cell lines: effects of cell-cell contact and nutritional deprivation.

Abstract Chandna, S., Dwarakanath, B. S., Khaitan, D., Mathew, T. L. and Jain, V. Low-Dose Radiation Hypersensitivity in Human Tumor Cell Lines: Effects of Cell–Cell Contact and Nutritional Deprivation. Radiat. Res. 157, 516–525 (2002). The hyper-radiosensitivity at low doses recently observed in vitro in a number of cell lines is thought to have important implications for improving tumor radiotherapy. However, cell–cell contact and the cellular environment influence cellular radiosensitivity at higher doses, and they may alter hyper-radiosensitivity in vivo. To confirm this supposition, we investigated the effects of cell density, multiplicity and nutritional deprivation on low-dose hypersensitivity in vitro. Cell survival in the low-dose range (3 cGy to 2 Gy) was studied in cells of two human glioma (BMG-1 and U-87) and two human oral squamous carcinoma (PECA-4451 and PECA-4197) lines using a conventional macrocolony assay. The effects of cell density, multiplicity and nutritional deprivation on hyper-radiosensitivity/induced radioresistance were studied in cells of the BMG-1 cell line, which showed prominent hypersensitivity and induced radioresistance. The induction of growth inhibition, cell cycle delay, micronuclei and apoptosis was also studied at the hyper-radiosensitivity-inducing low doses. Hyper-radiosensitivity/induced radioresistance was evident in the cells of all four cell lines to varying extents, with maximum sensitivity at 10–30 cGy, followed by an increase in survival up to 50 cGy–1 Gy. Both the glioma cell lines had more prominent hyper-radiosensitivity than the two squamous carcinoma cell lines. Low doses inducing maximum hyper-radiosensitivity did not cause significant growth inhibition, micronucleation or apoptosis in BMG-1 cells, but a transient G1/S-phase block was evident. Irradiating and incubating BMG-1 cells at high density for 0 or 4 h before plating, as well as irradiating cells as microcolonies, reduced hyper-radiosensitivity significantly, indicating the role of cell–cell contact-mediated processes. Liquid holding of BMG-1 cells in HBSS + 1% serum during and after irradiation for 4 h significantly reduced hyper-radiosensitivity, suggesting that hyper-radiosensitivity may be due partly to active damage fixation processes at low doses. Therefore, our findings suggest that the damage-induced signaling mechanisms influenced by (or mediated through) cell–cell contact or the cellular environment, as well as the lesion fixation processes, play an important role in hyper-radiosensitivity. Further studies are required to determine the exact nature of the damage that triggers these responses as well as for evaluating the potential of low-dose therapy.

Radiation responses of Sf9, a highly radioresistant lepidopteran insect cell line

Purpose: Lepidopteran insect cells are known to exhibit very high radioresistance. Although very effective DNA excision–repair has been proposed as a contributing factor, a detailed understanding of insect cell radiation responses has not yet been obtained. Therefore, the study was carried out to understand the in vitro radiation responses of Sf9 lepidopteran cells. Materials and methods: Exponentially growing asynchronous Sf9 cells (derived from ovaries of Spodoptera frugiperda) were exposed to gamma‐radiation doses of 2–200 Gy. Cell survival, growth inhibition, cell cycle progression delay, alterations in cell morphology as well as induction of DNA damage, micronuclei and apoptosis were studied at various post‐irradiation time intervals. Results: Biphasic survival response curves were obtained with D0 rising from 20 Gy (at doses ≤60 Gy) to 85 Gy (between 60 and 200 Gy), corroborating earlier reports on lepidopteran cells. An additional downward deviation at 2 Gy indicated a hypersensitive response. Dose‐dependent growth inhibition with a transient G2 delay starting 12 h and extending up to 48–96 h was observed at doses of 10–200 Gy, while a brief G1/S transition delay was observed only at higher doses (≥100 Gy). Significant DNA damage was detected only at 20 Gy and higher doses, in contrast with human cells that showed similar damage at 2 Gy. Interestingly, micronuclei were not induced at any of the doses tested, although spontaneous micronucleation was evident in <1% of cells. Lack of micronucleus induction even at doses that induced significant DNA damage and a transient G2 block (20–50 Gy) strongly indicated a role of holocentric lepidopteran chromosomes. Apoptosis was detected only in a small proportion of cells (3%) exposed to 200 Gy, and cell/nucleus size and granularity increased by 72–96 h post‐irradiation in a dose‐dependent manner. Sf9 nucleoids extracted at 2 M NaCl showed higher compactness than the nucleoids prepared from human cells. Conclusions: It is clearly shown that lepidopteran cells are highly resistant to the induction of DNA damage and micronuclei, and display very low induction of apoptosis at doses up to 200 Gy. While the lack of micronucleus induction seems to be primarily due to the holocentric nature of their chromosomes, certain unique signalling pathways might be responsible for the low induction of apoptosis. Factors causing protection of Sf9 cellular DNA from radiation‐induced damage are presently being investigated.

MODIFICATION OF THE RADIATION INDUCED DAMAGE BY 2-DEOXY- D-GLUCOSE IN ORGAN CULTURES OF HUMAN CEREBRAL GLIOMAS

Effects of a glucose antimetabolite, 2-deoxy-D-glucose (2-DG), on the gamma ray induced radiation damage have been studied in organ cultures of human cerebral gliomas. Percentage of cells with micronuclei (M-fraction) was used to assay the radiation damage. Experimental data indicate the following results. Untreated cerebral gliomas show considerable spatial heterogeneity in M-fraction; In spite of this heterogeneity, increases in M-fraction induced by gamma rays can be clearly observed, if multiple and randomly selected explants are analyzed for each group; The radiation induced M-fraction in different gliomas varies over a wide range; Presence of 2-DG (5 mM) for 4 h after irradiation leads to an increase in the radiation induced M-fraction in the majority of tumors, while in a smaller number (congruent to 25%) a decrease is observed under similar conditions. These results can be explained on the basis of a model postulating differential effects of 2-DG on the energy linked modulations of the processes of repair and fixation of DNA damage, which competitively influence the formation of micronuclei.

Cellular uptake, localization and photodynamic effects of haematoporphyrin derivative in human glioma and squamous carcinoma cell lines.

Uptake, intracellular concentration, localization and photodynamic effects of a haematoporphyrin derivative (HpD, Photosan-3) were compared in human glioma (BMG-1, wild-type p53) and squamous carcinoma (4451, mutated p53) cell lines. Concentration and time dependence of cellular uptake of HpD was assayed from methanol extracts and whole cell suspension spectroscopy, while localization was studied by fluorescence microscopy-based image analysis. Colony-forming ability, apoptosis, cell-cycle progression and cytogenetic damage (micronuclei formation) were investigated as parameters of photodynamic response following irradiation with red light. BMG-1 cells were more sensitive to the photodynamic treatment than 4451 cells, although the 4451 cells accumulated a higher amount of HpD and did not differ significantly from BMG-1 cells with respect to intracellular localization. Photodynamically-induced cytogenetic damage and apoptosis were considerably higher in BMG-1 cells as compared to 4451 cells. The present results strongly suggest that manifestation of the photodynamically-induced lesions in the form of cytogenetic damage and apoptosis are among the important determinants of cellular sensitivity to HpD-PDT besides the photodynamic dose (intracellular concentration of the photosensitizer and the light dose).

Hematoporphyrin derivatives potentiate the radiosensitizing effects of 2-deoxy-D-glucose in cancer cells

PURPOSE Two deoxy-D-glucose (2-DG), an inhibitor of glucose transport and glycolysis, has been shown to differentially inhibit the repair of radiation damage in cancer cells by reducing the flow of metabolic energy. Since hematoporphyrin derivatives (Hpd) inhibit certain enzymes of the respiratory metabolism, resulting in an increase in the glucose usage and glycolysis, Hpd could possibly enhance the energy-linked radiosensitizing effects of 2-DG in cancer cells. The purpose of the present work was to verify this suggestion. METHODS AND MATERIALS Two human tumor cell lines (cerebral glioma, BMG-1 and squamous cell carcinoma, 4197) and a murine tumor cell line (Ehrlich ascites tumor [EAT], F-15) in vitro were investigated. A commercially available preparation of Hpd, Photosan-3 (PS-3) was used in the present studies. Cells incubated with 0-10 microg/ml PS-3 for 0-24 h before irradiation were exposed to 2.5 Gy of Co-60 gamma rays and maintained under liquid holding conditions for 1-4 h to facilitate repair. 2-DG (0-5 mM) added at the time of irradiation was present during the liquid holding. Radiation-induced cytogenetic damage (micronuclei formation) and cell death (macrocolony assay) were analyzed as parameters of radiation response. Effects of these radiosensitizers on glucose usage and glycolysis were also studied by measuring the glucose consumption and lactate production using enzymatic assays. RESULTS The glucose consumption and lactate production of BMG-1 cells (0.83 and 1.43 pmole/cell/h) were twofold higher than in the 4197 cells (0.38 and 0.63 pmole/cell/h). Presence of PS-3 (10 microg/ml) enhanced the rate of glycolysis (glucose consumption and lactate production) in these cells by 35% to 65%, which was reduced by 20% to 40% in the presence of 5 mM 2-DG. In exponentially growing BMG-1 and EAT cells, presence of 2-DG (5 mM; equimolar with glucose) for 4 hours after irradiation increased the radiation-induced micronuclei formation and cell death by nearly 40%, whereas no significant effects could be observed in 4197 cells. In EAT cells, radiation was also observed to induce apoptotic death, which was significantly increased in the presence of the combination (PS-3 + 2-DG). The combination (PS-3 + 2-DG) enhanced the radiation damage in all three cell systems by 60-100%. Furthermore, the radiosensitizing effects of the combination (PS-3 + 2-DG) were higher at pH 6.7 as compared to pH 7. 4. In the plateau phase, presence of 2-DG alone did not significantly influence the radiation response of either BMG-1 or of 4197 cells, whereas in combination with PS-3, 2-DG enhanced the radiation damage in both these cell lines by 40% to 50%. Furthermore, in BMG-1 cells, the effects of 2-DG were observed to be reversible to a very great extent, while that of the combination were mostly irreversible. CONCLUSION The hematoporphyrin derivative PS-3 enhances the radiosensitizing effects of 2-DG in cancer cells, possibly by further reducing the energy supply leading to an irreversible inhibition of DNA repair, and increased cytogenetic damage and cell death. Since both these compounds have been used in clinical practice, further studies to investigate their use in improving radiotherapy of tumors are warranted.

In vitro and in vivo targeted delivery of photosensitizers to the tumor cells for enhanced photodynamic effects

BACKGROUND Efficacy of photodynamic therapy can be enhanced by improving uptake, localization, and sub-cellular localization of sensitizers at the sensitive targets. MATERIALS AND METHODS Uptake, localization, and photodynamic effects of hematoporphyrin derivative (HpD, Photosan-3; PS-3) and disulfonated aluminum phthalocyanine (AlPcS₂) were studied either encapsulated in liposomes or conjugated to a monoclonal antibody to carcinoembryonic antigen (anti-CEA) in a brain glioma cell line, BMG-1. RESULTS Although the total uptake with encapsulated or conjugated sensitizers was less than the free sensitizers, photodynamic efficiency was higher due to the localization of the sensitizer at the sensitive targets. Biodistribution of intravenously administered technetium (⁹⁹m Tc)-labeled PS-3 analyzed by gamma camera imaging showed maximum accumulation in the liver followed by tumor. Tumor/muscle (T/N) ratio of free PS-3 was higher compared to encapsulated or conjugated PS-3 but the accumulation of PS-3 significantly reduced in brain and cutaneous tissue following modulated delivery. Pharmacokinetics suggested faster accumulation of encapsulated and conjugated PS-3 in the tumor. CONCLUSION Localization of sensitizers at sensitive targets and reduced accumulation in normal tissues with liposome encapsulation and antibody conjugation suggest that these two delivery systems can potentially enhance the efficacy of photodynamic treatment.