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Plasmin-Depletion Therapy

A New Approach to Overcoming Tumor Cell Drug Resistance

MYUNG CHUNa,c AND MICHAEL K. HOFFMANNb

aBioeast, 200 East End Avenue, New York, New York 10128, USAbNew York Medical College, Valhalla, New York, New York 10595, USA

Most anticancer agents destroy cancer cells by activating the apoptosis pathway.1

We have shown that plasmin can render cancer cells resistant to apoptosis-inducingdrugs and may alter their growth pattern.2,3

The adverse effect of plasmin on the growth of cancer has been recognized. Theprecursor of plasmin is plasminogen. Cancer cells often express the protease uroki-nase, which converts plasminogen to plasmin. Urokinase has been detected in breastcancer, colorectal cancer, lung cancer, melanoma, prostate cancer, stomach cancer,and ovarian cancer.4–6 Plasmin generation by urokinase in tumor tissues is known tocorrelate with a poor prognosis.4–6 In the last decade, extensive but unsuccessful at-tempts have been made to inhibit the generation of plasmin on cancer cells by block-ing tumor cell urokinase.4–6

We have taken a different approach. We blocked plasmin generation on cancercells by eliminating the urokinase substrate plasminogen rather than by inhibitingthe enzyme itself. This therapeutic approach is referred to as plasmin-depletion ther-apy. The plasmin-depletion therapy principle is illustrated in FIGURE 1.

Plasmin-depletion therapy induces systemic conversion of the patient’s plasmi-nogen to plasmin. On conversion, plasmin is rapidly inactivated by decay. As it takesapproximately 2 days for the body to regenerate plasminogen, plasmin-depletiontherapy provides a plasminogen-free state in patients and deprives cancer cells of theplasmin precursor plasminogen. During this period, cancer cells are unable to blockthe apoptosis process and remain sensitive to apoptosis-inducing drugs (FIG. 1).

Preclinical data show that plasmin-depletion therapy enhances the efficacy ofanticancer agents and of the host’s anticancer defense. Tissue culture studies wereperformed with several apoptosis-inducing anticancer agents and a variety of humanbreast and colon cancer cell lines. The following are examples of in vivo experi-ments. Immunodeficient nu/nu mice were inoculated intradermally with HT29 hu-man colon cancer cells. One day later, the animals received saline as control, PDT,a suboptimal dose of doxorubicin, or doxorubicin plus PDT. Tumor volumes wereassessed at 21 days. The volume of the tumors in the group treated with doxorubicinplus PDT was less than half that in the group treated with either doxorubicin or PDTalone. Tumor volume in the latter two groups was approximately 85% that of thecontrol group. In immunotherapies, similar results were obtained. Fourteen days af-

cFor correspondence: phone, 212/360-6631; fax, 212/360-6631.e-mail, chun01@sprynet.com

241CHUN & HOFFMANN: PLASMIN-DEPLETION THERAPY

FIGURE 1. Plasmin-deple-tion therapy principle. Untreat-ed: Plasmin generated frompatient’s plasminogen by uroki-nase induces drug resistance.PDT treated: Plasmin-depletiontherapy depletes patient’s plas-minogen, the source of plasmingenerated by cancer cells, andthus suppresses drug resistance.

FIGURE 2. Plasmin-depletion therapy increases the efficacy of chemotherapeutic andimmunotherapeutic drugs in vivo. (A) Balb/C athymic nude mice were injected intraperito-neally with either 0.2 ml saline (�,�) or plasmin-depletion therapy (PDT, 150 units uroki-nase in 0.2 ml saline) (�,�). Thirty minutes later, mice were injected with saline solutionor PDT twice more at 10-minute intervals. Two hours after the first injection, 2 millionHT29 cells were inoculated intradermally into the abdomen. Mice were treated twice, 24 and48 hours after tumor inoculation, with 0.2 ml saline (�,�) or 100 µg/ml M79 antibody in

242 ANNALS NEW YORK ACADEMY OF SCIENCES

ter treatment, tumor volume in the group treated with monoclonal tumor-reactive an-tibody was 50% that of the control group. Tumor volume in the animals receivingPDT alone was 80% of control. In animals treated with monoclonal antibody plusPDT, tumors were not detected (FIG. 2).

In conclusion, these observations indicate that plasmin-depletion therapy, byovercoming a key form of drug resistance, provides a means to increase the efficacyof systemic therapies.

REFERENCES

1. da SILVA C.P., C.R. DE OLIVEIRA, M. DA CONCICAO & P. DE LIMA. 1996. Apoptosis asa mechanism of cell death induced by different chemotherapeutic drugs in humanleukemic T- lymphocytes. Biochem Pharmacol. 51:1331–1340.

2. CHUN, M. 1997. Plasmin induces the formation of multicellular spheroids of breastcancer cells. Cancer Lett. 117: 51–56.

3. CHUN, M. & M.K.HOFFMANN. 1997. Patent application. PCT/US97/14231. Date ofApplication: August 13, 1997.

4. BACHMANN, F. 1994. Basic Principles and Clinical Practice. Lippincott. Co. Philadel-phia, PA.

5. FAZIOLI, F. & F. BLASI. 1994. Urokinase-type plasminogen activator and its receptor:New targets for anti-metastatic therapy? Trends Pharmacol. Sci 15: 25–29.

6. ANDREASEN, P.A., L. KJOLLER, L. CHRISTENSEN & M.J. DUFFY. 1997. The urokinase-type plasminogen activator system in cancer metastases: A review. Int. J. Cancer 72:1–22.

FIGURE 2 (continued from previous page)0.2 ml saline (�,�). On the days indicated, the formation of tumors as well as the volumeof tumors was assessed. Tumor volume = the length � the width � the height. (B) One mil-lion HT29 cells were inoculated into the abdomen of nude mice. Twenty hours later, animalswere treated with either plasmin-depletion therapy (i.e., 150 units urokinase) (�,�) or sa-line (�,�) twice at 30-minute intervals. After 24 hours, animals were treated with either0.2 ml saline (�,�) or 50 µg doxorubicin in 0.2 ml saline (�,�) intravenously twice at 4-hour intervals. Tumors were measured as described in A on the days indicated.