Tapas K. Das Gupta

Benign solitary schwannomas (neurilemomas)

Three hundred and three patients with benign solitary neurilemomas, or schwannomas, not associated with the classical type of von Recklinghausens disease were studied. One hundred and seventy‐two were women and 131 were men. Forty‐five percent of the tumors were seen in the extremities, and no specific area could be related to the development of these tumors. Approximately 9% of the tumors occurred in the soft tissues of the trunk and 14% in various unusual sites. Most patients were seen initially because of a painless mass; only 10 complained of pain radiating along the course of a peripheral nerve. One documented case of a malignant degeneration in a benign schwannoma has been reported. The treatment of a benign solitary neurilemoma is enucleation. Excision of a segment of a major peripheral nerve is contraindicated. The histologic features of benign peripheral nerve tumors were briefly described. The confusion regarding terminology has been discussed, and it is suggested that the term schwannoma be more frequently used. The lack of neurologic deficit in schwannomas arising in major peripheral nerves has been pointed out, and further studies have been suggested. The high association of malignant tumors in these patients has been discussed, and it is proposed that a further study be undertaken to ascertain whether these patients are more prone to develop unrelated cancers.

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P=0.0179, Kruskal–Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

Soft tissue sarcomas of the adult head and neck

From 1969 to 1983, 53 adult patients with head and neck soft tissue sarcomas were evaluated and treated by the Division of Surgical Oncology at the University of Illinois. The most common anatomic location was the neck (36%), and these patients had the highest 5‐year disease‐free survival rate (67%). Fibrosarcoma was the most common histologic type (26%); patients with aggressive fibromatosis had the longest mean survival time (93 months). The mean overall survival time was 58.7 months, and the disease‐free 2‐year, 5‐year, and 10‐year survival rates were 68%, 54%, and 28%, respectively. Wide excision was the treatment of choice, with adjuvant radiotherapy or chemotherapy, or both, used in selected patients. In all of the long‐term survivors, the tumors were either well‐differentiated or ≤5.0 cm in diameter. It is apparent that aggressive therapy of such tumors can provide good long‐term results.

A Phase I Trial of TNFerade Biologic in Patients with Soft Tissue Sarcoma in the Extremities

Purpose: TNFerade is a second-generation replication-deficient adenovector carrying a transgene encoding human tumor necrosis factor α under control of a radiation- induced promoter. The objective of this study was to assess the tolerance of combining TNFerade and radiation therapy in patients with soft tissue sarcomas of the extremity. Experimental Design: TNFerade was administered in combination with single-daily fractionated radiation therapy in 14 patients with soft tissue sarcoma of the extremities. Three escalating dose levels of TNFerade (4 × 109 −4 × 1011 particle units) were planned, given in 1 log increments by intratumoral injections, twice weekly during week 1 and once weekly during weeks 2–5 of radiation therapy. Results: TNFerade was well tolerated with no dose-limiting toxicities noted. Grade 1–2 chills (50.0%), fever (43.0%), fatigue (36.0%), and flu-like symptoms (21.0%) were the most common side effects. Serum-tumor necrosis factor α levels were low in all of the patients (<15 pg/mL). No patients had virus-detected blood, sputum, or urine cultures. Of the 13 evaluable patients, 11 received TNFerade preoperatively, and 2 received the treatment for palliation. Eleven patients (85%) showed objective or pathological tumor responses (2 complete and 9 partial), and 1 had stable disease. Partial responses were achieved despite some of these tumors being very large (up to 675 cm2). Of the 11 patients who underwent surgery, 10 (91%) showed a pathological complete response/partial response. Conclusion: TNFerade + radiation therapy was well tolerated in the treatment of patients with soft-tissue sarcoma of the extremity. The high number of objective responses observed warrants additional studies of this approach in a larger controlled prospective trial.

Steroid receptors in human lung cancer cytosols

Receptors for all classes of steroid hormones were identified in cytosols of adenocarcinoma of the lung. In contrast, the incidence of receptor in squamous cell carcinoma was limited and small cell carcinomas appeared devoid of receptor. Receptor binding was of high affinity, saturable and localized to the 8, 6-7 and 4s regions of sucrose density gradients. Demonstration of high affinity cytosol receptors for steroids in adenocarcinoma and squamous cell carcinoma of the lung provides suggestive initial evidence that steroids may influence the natural history of a subset of human lung carcinomas.

Blood and lymph vessel tumors

A soft tissue sarcoma arising in the blood and/or lymph vessels is relatively rare; yet, when it occurs, this type of tumor may present a challenge to the physician. Since the vast majority of such sarcomas are benign blood vessel hemangiomas, which seldom require any treatment, when a true malignant hemangioendothelioma is encountered, the physician may be at a loss in deciding on either management or prognosis. To counter this, some general findings in blood and lymph vessel tumors are dis cussed below. Remember, however, that none of these sarcomas can be posi tively identified without proper histolog ical examination—the essence of good management.

Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

Molecular prognostic markers in intermediate-thickness cutaneous malignant melanoma

The limitations of morphologic criteria alone in determining the prognosis for a patient with a particular intermediate‐thickness primary melanoma have prompted efforts to identify other markers.

Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity.

Azurin is a member of a group of copper‐containing redox proteins called cupredoxins. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recently, we demonstrated that azurin enters into J774 and several types of cancer cells leading to the induction of apoptosis. We now demonstrate that azurin is internalized in J774 or cancer cells in a temperature‐dependent manner. Azurin shows preferential entry into cancer compared with normal cells. An 28‐amino‐acid fragment of azurin fused to glutathione S‐transferase (GST) or the green fluorescent protein (GFP), which are incapable of entering mammalian cells by themselves, can be internalized in J774 or human melanoma or breast cancer cells at 37°C, but not at 4°C. Competition experiments as well as studies with inhibitors such as cytochalasin D suggest that azurin may enter cells, at least in part, by a receptor‐mediated endocytic process. The 28‐amino‐acid peptide therefore acts as a potential protein transduction domain (PTD), and can be used as a vehicle to transport cargo proteins such as GST and GST–GFP fusion proteins. Another member of the cupredoxin family, rusticyanin, that has also been shown to enter J774 and human cancer cells and exert cytotoxicity, does not demonstrate preferential entry for cancer cells and lacks the structural features characteristic of the azurin PTD.