Marie Selzer

Inhibition of cell proliferation, invasion, tumor growth and metastasis by an oral non-antimicrobial tetracycline analog (COL-3) in a metastatic prostate cancer model

Antibiotic forms of tetracycline exhibit antitumor activity in some tumor models. However, their low in vivo efficacy and associated morbidity limit their long‐term application in cancer therapy. This report appraises the efficacy of doxycycline (DC) and non‐antimicrobial, chemically modified tetracyclines (CMTs) against prostate cancer. Both DC and several CMTs inhibited prostate tumor cell proliferation in vitro. Some of the CMTs were significantly more potent than DC. One of the CMTs, 6‐deoxy, 6‐demethyl, 4‐de‐dimethylamino tetracycline (CMT‐3, COL‐3), was the most potent inhibitor (50% inhibition dose [GI50] ≤ 5.0 μg/ml). Exposure of tumor cells to CMT‐3 induced both apoptosis and necrosis. Mitochondrial depolarization and increased levels of reactive hydroxyl radicals were also observed in cells treated with CMT‐3. Cell cycle arrest at the G0/G1 compartment was observed in CMT‐3‐ and DC‐treated cells. DC and CMTs also inhibited the invasive potential of the tumor cells in vitro, from 10% (CMT‐6) to >90% (CMT‐3). CMT‐3 and DC decreased matrix metalloproteinase (MMP)‐2, tissue inhibitor of MMP (TIMP)‐1 and TIMP‐2 secretion in treated cultures and inhibited activity of secreted MMPs, CMT‐3 was a stronger inhibitor. Daily oral gavage of DC and CMT‐3 inhibited tumor growth and metastasis in the Dunning MAT LyLu rat prostate tumor. Decreases in tumor growth (27–35%) and lung metastases were observed (28.9 ± 15.4 sites/animal [CMT‐3‐treated] versus 43.6 ± 18.8 sites/animal [DC‐treated] versus 59.5 ± 13.9 [control]; p < 0.01]. A delay in tumor growth (27 ± 9.3%, p < 0.05), reduction in metastases (58 ± 8%) and decrease in tumor incidences (55 ± 9%, CMT‐3‐treated) were also observed, when rats were predosed for 7 days. No significant drug‐induced morbidity was observed in any of the animals. These results, along with a recently concluded clinical trial, suggest a potential use of CMT‐3 as an oral, nontoxic drug to treat metastatic prostate and other cancers.

THE ASSOCIATION OF ELEVATED URINARY TOTAL TO SULFATED GLYCOSAMINOGLYCAN RATIO AND HIGH MOLECULAR MASS HYALURONIC ACID WITH INTERSTITIAL CYSTITIS

PURPOSE A decrease in the glycosaminoglycan (GAG) layer on the urothelium is believed to be one of the possible causes of interstitial cystitis. Consequently, GAG-like substances and hyaluronic acid (HA) have been prescribed for treating this condition. To delineate the possible role of GAG and HA in the interstitial cystitis disease process, we compared the urinary levels of total GAGs (sulfated + non-sulfated), sulfated GAGs and HA in interstitial cystitis patients and normal controls. We also examined different HA species present in the urine of interstitial cystitis patients. MATERIALS AND METHODS The total GAG and sulfated GAG levels in urine specimens of normal individuals (n = 20) and interstitial cystitis patients (n = 25) were determined by utilizing the carbazole reaction assay and the Farndale method, respectively, and were expressed as microg./mg. creatinine. Urinary HA levels were measured by applying the HA test and were expressed as ng./mg. creatinine. Gel filtration column chromatography was used to examine the profile of urinary GAGs and HA species. RESULTS Total urinary GAGs were 2.5 to 4-fold elevated in interstitial cystitis patients with moderate to severe symptoms (Group 2; 76.2 +/- 24.8) when compared with those in normal individuals (19.9 +/- 2.5) and patients with mild symptoms (Group 1; 30.4 +/- 5.1) (p <0.001). Three urinary GAG peaks were detected in both normal and interstitial patients. However, each GAG peak from interstitial cystitis patient urine was 3 to 5-fold higher than that from normal patient urine. The sulfated GAG levels, however, remained unchanged among normal individuals (1.4 +/- 0.22), Group 1 (2.2 +/- 0.96) and Group 2 (1.6 +/- 0.38) patients (p >0.05). Consequently, the ratio of total GAGs to sulfated GAGs was elevated 3 to 3.5-fold in Group 2 patients (49.9 +/- 13.9) in comparison to that in normal individuals (16.7 +/- 2.5) and group 1 patients (14.4 +/- 4.6) (p <0.001). Urinary HA levels were marginally elevated in Group 2 patients (821. 4 +/- 247.9) when compared with those in the normal group (337.3 +/- 106.1) and Group 1 patients (540.9 +/- 166.5). In addition, a distinct high molecular mass HA species was present only in Group 2 patients. CONCLUSIONS The increased ratio of total GAGs to sulfated GAGs and marginally elevated HA levels in urine indicate that the GAG layer is altered in interstitial cystitis patients. However, these results are in contrast to the accepted concept that a reduction in urothelial GAGs causes interstitial cystitis. The high molecular mass HA species detected in patients with severe symptoms may play a role in the pathophysiology of this disease.

CMT-3, a Chemically Modified Tetracycline, Inhibits Bony Metastases and Delays the Development of Paraplegia in a Rat Model of Prostate Cancer

In 1998 an estimated 186,500 Americans will be diagnosed with prostate cancer. 1 At the time of diagnosis approximately 50% of prostate cancer patients will have some form of extraprostatic disease—e.g., metastasis to lymph node, lung, and bone. 2 Carcinoma of the prostate is, by far, the most common of the neoplasms that produce osteoblastic metastases. 3 These metastases result from tumor cell invasion into the bony matrix by the enzymatic degradation of collagen and other matrix components. 4 Key enzymes in collagen degradation are the matrix metalloproteinases (MMPs), secreted by tumor and stromal cells, during tumor-induced bone remodeling. 5 Existing therapies to control this painful disease are only palliative, not curative. 6 We reported, some years ago, that primary cultures of human prostate tissue secrete high levels of activated MMP-2 and MMP-9, but low to undetectable amounts of their natural endogenous inhibitors (TIMPs). 7 In this study we examined whether CMT-3 (6-demethyl, 6-deoxy, 4-dedimethylamino tetracycline) a nontoxic, non-antimicrobial, orally bioavailable MMP inhibitor, with a strong affinity to bone, 8 could inhibit prostate cancer skeletal metastasis.

Potential application of a chemically modified non-antimicrobial tetracycline (CMT-3) against metastatic prostate cancer.

The matrix metalloproteinases (MMPs) are known to play a significant role in tumor cell invasion and metastasis (Liotta and Stetler-Stevenson, 1990; Stetler-Stevenson et al., 1993; Chambers et al, 1995; Lawrence and Steeg, 1996). Typically, aggressive tumor cells secrete a large excess of MMPs and low levels of endogenous inhibitors (e.g., TIMPs) (Liotta et al, 1991). This imbalance is known to be a causative factor in invasion and metastasis (Liotta et al., 1991; Lokeshwar et al, 1993). Recently, several synthetic inhibitors of MMPs have been tested for their potential to prevent or reduce tumor metastases (Davies et al, 1993; Brown, 1995; Sledge et al, 1995; Wojtowicz-Praga et al, 1997). Several of these inhibitors have limited effectiveness as therapeutic agents due to poor solubility (Davies et al, 1993), lack of oral availability (Brown, 1995), or toxicity (Sledge et al, 1995). An orally administrable, non-toxic inhibitor which neutralizes the MMPs would be a prime therapeutic candidate. Cytotoxicity to proliferating cells, with little systemic toxicity, would be additional virtues of such a drug. Tetracyclines such as doxycycline (DC) and several chemically modified non-antimicrobial tetracyclines (CMTs)

Hyaluronic Acid and Hyaluronidase: Accurate Tumor Markers and Tumor Progression Regulators

INTRODUCTION. Identification of molecular determinants that regulate tumor progression would help in improving diagnosis and prognosis for cancer patients. Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan that promotes tumor metastasis. Hyaluronidase (HAase) is an endoglycosidase that degrades HA into small angiogenic fragments. In an earlier study we demonstrated that measurement of urinary levels of HA and HAase (HA-HAase test) is > 90% accurate in detecting bladder cancer (BCa) and evaluating its grade. In this study, we examined the ability of the HA-HAase test to monitor patients for BCa recurrence and examined the effect of HA fragments, isolated from patient samples, on endothelial cell functions.

CHAPTER 11 – Hyaluronidase: Both a Tumor Promoter and Suppressor

Publisher Summary Hyaluronidases (HAases) are a class of enzymes that predominantly degrade hyaluronic acid (HA) and are present in a variety of toxins and venoms. Despite a lot of work on bacterial, invertebrate, and testicular HAases, a connection between HAase and cancer was unequivocally established just over a decade ago and the functional significance of HAases in cancer was demonstrated. This chapter concerns the recent advances in the understanding of the role of HAases in cancer. Various studies have established a link between HAase and the tumor invasive/metastatic phenotype.HAase expression appears to be elevated in many carcinomas and the expression correlates with tumor invasiveness. However, in some carcinomas HAase expression depends on the status of the chromosome 3p21.3 locus and may inversely correlate with tumor grade. The origin of the concept that HAeses are tumor suppressors lies in the observation that in some epithelial carcinomas, the 3p21.3 locus is deleted. The diagnostic potential of HAase, either alone or together with HA, has been extensively explored in bladder cancer. Standard clinical and pathological parameters provide very limited information to clinicians regarding which prostate cancers will progress and/or have a poor prognosis.