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The approach to the management of hyperparathyroidism during pregnancy varies, depending on the presence or absence of symptoms and their severity; the gestational age; and the patient’s preference. Conservative management with watchful waiting is often most reasonable for patients with mild, asymptomatic hypercalcemia (i.e., calcium levels that are slightly about the normal range for pregnancy). Increased oral intake of salt and fluids is recommended to prevent volume depletion. In more severe cases, intravenous hydration with isotonic saline is warranted; furosemide promotes urinary calcium excretion and may help in the treatment of patients with initial hypercalcemia, but it should be used only after volume repletion. Calcitonin, which is classified by the Food and Drug Administration as a category C medication for pregnant patients (i.e., a medication for which animal studies have shown an adverse effect on the fetus, but no adequate, well-controlled studies have been conducted in humans; potential benefits may warrant use of the drug in pregnant women despite potential risks), may bring about rapid reductions in calcium levels when administered intravenously or intramuscularly, but it is not a viable option for prolonged treatment, since tachyphylaxis rapidly develops. Bisphosphonates cross the placenta and are contraindicated in pregnancy owing to concern about their interference with fetal bone development.

Parathyroidectomy is the only definitive therapy and is generally recommended for cases of symptomatic and severe hypercalcemia. The second trimester is generally preferred for surgery, but for patients in whom medical management is ineffective, surgical intervention may be necessary irrespective of the stage of gestation. In all cases of maternal hyperparathyroidism, neonates should be followed closely for evidence of hypocalcemia resulting from suppression of PTH production by the neonatal parathyroid gland, which may not appear until several hours after delivery.

This case underscores the need to consider a broad differential diagnosis for problems in pregnancy and to interpret laboratory tests in the context of the complex metabolic alterations associated with pregnancy. In this patient, back and abdominal pain proved to be attributable to pancreatitis, which was probably caused by hypercalcemia associated with hyperparathyroidism. The detection of hypercalcemia and an inappropriately “normal” intact PTH level led to the identification of primary hyperparathyroidism, and surgical intervention in the second trimester resulted in a good outcome for both mother and infant.

An association between hyperparathyroidism and an increased prevalence of hypertension has been reported in nonpregnant patients, but the mechanism has not been identified. The effects of parathyroidectomy on blood-pressure levels in such patients have been inconsistent.

Commentary

Primary hyperparathyroidism occurs rarely during pregnancy — the true incidence is unknown. Since many cases are asymptomatic, they are not recognized in pregnant patients. In addition, pregnancy is associated with alterations in the levels of calcium and calcitropic hormones such as PTH, which may obscure the hyperparathyroidism. In this case, the presence of hypercalcemia was an important clue — a finding that was in clear contrast to the reduction in total serum calcium levels expected in pregnancy.

The morbidity associated with primary hyperparathyroidism during pregnancy is substantial, with complications reported in up to 67% of affected mothers and 80% of fetuses and neonates, usually in the presence of severe hypercalcemia (an increase in calcium levels of approximately 2 mg per deciliter [0.5 mmol per liter] or more above the normal range for pregnancy). Fetal complications associated with maternal hyperparathyroidism include restriction of intrauterine growth, low birth weight, preterm delivery, stillbirth, miscarriage, and neonatal tetany. The maternal complications are similar to those seen outside of pregnancy, including nephrolithiasis, pancreatitis, bone disease, changes in mental status, and hypercalcemic crisis. Pancreatitis during pregnancy is rare, occurring in 0.03% of pregnancies.8 Whereas some reports — most of them based on case series — have suggested an association between primary hyperparathyroidism and pancreatitis, a community-based study showed no increase in the incidence of pancreatitis among patients with primary hyperparathyroidism as compared with matched controls.

An understanding of the normal pregnancy-induced alterations in levels of calcium and vitamin D is important in the assessment of a patient with hypercalcemia in pregnancy. During pregnancy, calcium is shunted from the maternal circulation to the fetus, in order to mineralize the developing fetal skeleton. The fetal calcium demand increases primarily in the third trimester, but the maternal adaptations to meet this demand start early in pregnancy. Maternal shunting of calcium to the fetus may contribute to relative maternal hypocalcemia, but the reduction in total maternal serum calcium levels observed in pregnancy is mainly a reflection of a decrease in serum albumin levels and, consequently, a decrease in the albumin-bound fraction of calcium; ionized calcium levels remain in the normal range during pregnancy. Longitudinal measurements of intact PTH levels have revealed decreases to the low-to-normal range during early pregnancy, with a subsequent increase to the midnormal range by term.

A major maternal adaptation to the increased fetal calcium demand is increased intestinal absorption of calcium, mediated by an increase in 1,25-dihydroxyvitamin D levels. Maternal 1,25-dihydroxyvitamin D levels increase early in pregnancy and remain about twice as high as prepregnancy levels throughout pregnancy. The increase is attributed to PTH-independent up-regulation of 1α-hydroxylase in the maternal kidneys, with the placenta, decidua, and fetal kidneys possibly contributing additional amounts. The glomerular filtration rate also increases during pregnancy, as does urinary calcium excretion; the increased calcium excretion is probably a response to the increased intestinal absorption of calcium. Urinary calcium levels are commonly two to three times as high as they are in nonpregnant women and may be in a range that would be frankly hypercalciuric for nonpregnant women. PTH-related protein stimulates placental calcium transport in the fetus14 and may also have a role in protecting the maternal skeleton. Calcitonin may play a role in protecting the maternal skeleton from increased resorption.

Instructions were given for the patient to receive nothing by mouth, and she was treated with intravenous hydration, furosemide, and nasally administered calcitonin, with improvement in her calcium levels to a range of 1.32 to 1.38 mmol of ionized calcium per liter. Her pain subsided. Levels of amylase and lipase gradually fell. Methyldopa was discontinued, since pancreatitis is a rare side effect of this treatment. Treatment with oral labetalol was started for blood-pressure control. Fetal well-being was monitored by tracking the fetal heart rate, performing daily ultrasound examinations, and checking biophysical profiles, which remained normal.

It is likely that the patient’s hyperparathyroidism led to her pancreatitis. Her history of nephrolithiasis suggests that hypercalcemia may have been present for years. Although her symptoms have subsided and her calcium levels have improved, she continues to have mild hypercalcemia and is at risk for worsening hypercalcemia and attendant pregnancy-associated complications, including intrauterine growth retardation, preterm delivery, intrauterine fetal death, and neonatal tetany. Consequently, surgery should be considered in this patient. Preoperative imaging for identification and localization of an adenoma will reduce the duration and invasiveness of surgery. Since sestamibi scanning is contraindicated during pregnancy, ultrasonography of the neck would be the preferred imaging technique. After further abatement of the pancreatitis, when the patient could tolerate oral intake, she underwent parathyroid exploratory surgery and resection of a left lower parathyroid adenoma under general anesthesia, without complications. Blood samples were obtained from the internal jugular vein before and 10 minutes after resection of the adenoma, and analysis showed that the PTH level decreased from 344 to 60.7 pg per milliliter.
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The majority of cases of primary hyperparathyroidism are caused by solitary parathyroid adenomas. Intraoperative PTH monitoring takes advantage of the short half-life of PTH in plasma (3 to 4 minutes) and the availability of a rapid assay for PTH. A reduction in PTH levels of more than 50% is considered to be an indicator of successful removal of the adenoma. Postoperative monitoring of this patient’s serum calcium levels should be continued.

After resection, the patient’s serum calcium levels normalized and remained normal for the duration of her pregnancy. Maintenance doses of 600 mg of calcium carbonate with 200 IU of vitamin D twice daily were prescribed, in addition to 400 IU of vitamin D3 daily. The amylase and lipase levels gradually normalized. The patient remained in the hospital for continued monitoring because of mild preeclampsia. Labor was induced at 37.5 weeks’ gestation, after her blood pressure rose further, to 152/104 mm Hg, despite the administration of labetalol. She delivered a healthy girl by means of cesarean section, which was performed because of the failure of labor to progress. The postpartum serum calcium levels remained normal. After a course of 50,000 units of ergocalciferol weekly for 8 weeks, the 25-hydroxyvitamin D level increased to 30 ng per milliliter. The patient had persistent postpartum hypertension, requiring the continuation of antihypertensive therapy, but was otherwise well.

This patient has a high calcium level, which may be the cause of her pancreatitis. This finding is particularly notable because total calcium levels are lower in normal pregnancy than in the nonpregnant state. In contrast, levels of ionized calcium remain unchanged throughout pregnancy. Evaluation is warranted for hyperparathyroidism, since this is the most common cause of hypercalcemia. Levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D should also be measured to rule out the possibility of vitamin D intoxication.

The patient’s medical history was notable for pregnancy-induced hypertension and anemia, which were diagnosed at approximately 27 weeks’ gestation. Four years before presentation she had a kidney stone, which passed spontaneously; stone analysis was not performed. Her medications included methyldopa (250 mg twice daily) and a prenatal vitamin daily. She did not smoke, drank two glasses of wine per week before becoming pregnant and none during pregnancy, and had no history of illicit-drug use. Her family history was notable for type 2 diabetes mellitus in her parents and paternal grandmother, prostate cancer in her father, and breast cancer in two paternal aunts.

The ionized calcium level was 1.42 mmol per liter (reference range, 1.13 to 1.32). The level of intact parathyroid hormone (PTH), measured on the night of admission, when the serum calcium level was 11.1 mg per deciliter (2.8 mmol per liter), was 52.6 pg per milliliter (reference range, 11 to 80). Intravenous hydration was administered, with morphine given as needed for pain control. The next day, the serum calcium level was 10.1 mg per deciliter (2.5 mmol per liter), and the intact PTH level 85.4 pg per milliliter; the phosphorus level was 2.3 mg per deciliter (reference range, 2.4 to 5.0). The thyrotropin level was 1.35 mIU per liter, 25-hydroxyvitamin D 12 ng per milliliter (reference range, 30 to 60), and 1,25-dihydroxyvitamin D 96 pg per milliliter (reference range, 15 to 75).

The laboratory data are consistent with primary hyperparathyroidism. Whereas the initial level of intact PTH was within the normal range, it is high given the elevated calcium level. PTH levels are typically in the low-to-midnormal range during pregnancy. The patient’s 25-hydroxyvitamin D level is low, whereas her 1,25-dihydroxyvitamin D level is elevated. Although an elevated level of 1,25-dihydroxyvitamin D is a recognized cause of hypercalcemia in nonpregnant patients with certain neoplastic or granulomatous disorders (e.g., lymphoma, sarcoidosis, or tuberculosis), in this patient, the elevated level may simply reflect the physiologic increase in 1,25-dihydroxyvitamin D during pregnancy. Elevated 1,25-dihydroxyvitamin D levels are also observed in hyperparathyroidism as a result of increased conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. This increased conversion might also explain the patient’s low level of 25-hydroxyvitamin D, although the level is low enough to suggest concomitant vitamin D deficiency. If she has vitamin D deficiency, it could be keeping her serum calcium level less elevated than it otherwise would be. This combination of factors in this patient indicated that caution should be used during vitamin D repletion.

The white-cell count was 11,700 per cubic millimeter, the hematocrit 29.7%, and the platelet count 275,000 per cubic millimeter. The serum level of sodium was 135 mmol per liter, potassium 4.3 mmol per liter, chloride 105 mmol per liter, bicarbonate 21 mmol per liter, creatinine 0.7 mg per deciliter (61.8 μmol per liter), and glucose 70 mg per deciliter (3.9 mmol per liter). The calcium level was 11.4 mg per deciliter (2.8 mmol per liter) (reference range, 8.8 to 10.5 mg per deciliter), and the albumin level 3.4 g per deciliter (reference range, 3.6 to 4.6).

The level of alanine aminotransferase was 18 U per liter (reference range, 7 to 40), aspartate aminotransferase 45 U per liter (reference range, 6 to 40), alkaline phosphatase 159 U per liter (reference range, 27 to 110), total bilirubin 0.3 mg per deciliter (5.1 μmol per liter) (reference range, 0.3 to 1.2 mg per deciliter [5.1 to 20.5 μmol per liter]), amylase 617 U per liter (reference range, 20 to 70), and lipase 1261 U per liter (reference range, 3 to 60). Ultrasonography of the right upper quadrant of the abdomen revealed no evidence of gallstones or cholecystitis. The liver and common and intrahepatic bile ducts appeared normal. The pancreas was obscured by bowel gas.
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The patient was transferred to a tertiary-care hospital with a diagnosis of acute pancreatitis. At the time of admission, her blood pressure was 126/80 mm Hg. She was pain-free and drowsy after having received morphine in the emergency room, and her abdomen was not tender. Repeat laboratory testing revealed serum levels of calcium of 11.8 mg per deciliter (3.0 mmol per liter), albumin 3.3 g per deciliter, triglycerides 200 mg per deciliter (2.2 mmol per liter), lipase 2008 U per liter, and amylase 833 U per liter. Magnetic resonance imaging (MRI) of the abdomen revealed a diffusely enlarged, edematous, heterogeneous-appearing pancreas with small amounts of peripancreatic stranding and fluid, findings that were consistent with acute pancreatitis.

MRI Scan of the Abdomen Obtained without the Administration of Contrast Material. stranding and fluid, findings that were consistent with acute pancreatitis Magnetic resonance cholangiopancreatography revealed a normal-appearing gallbladder and no dilatation of the biliary or pancreatic ducts.

The two most common causes of acute pancreatitis in adults are gallstones and alcoholism. Less common causes include certain drugs (e.g., didanosine and valproic acid), hypertriglyceridemia, hypercalcemia, infection, trauma, ischemia, and conditions causing ampullary obstruction, such as periampullary diverticula or pancreatic or periampullary tumors; there are also rare inherited forms of pancreatitis. Pancreatitis is uncommon in pregnancy, and when it does occur, the cause is most often of biliary origin. Plasma triglyceride levels are increased by a factor of two to four during pregnancy, a change that is inconsequential in most pregnant women but that may result, in the presence of an underlying lipid disorder, in severe hypertriglyceridemia, precipitating pancreatitis; however, the triglyceride level is normal in this patient.

A 39-year-old woman (gravida 2, para 0) presented to her obstetrician at 32 weeks’ gestation with a 2-day history of low back pain. The pain was abrupt in onset and constant. She reported no fever, chills, dysuria, urinary frequency, vaginal discharge or bleeding, or other associated symptoms. Preterm labor was ruled out, and she was advised to rest and take acetaminophen as needed.
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All the possible causes of low back pain in women must be considered during pregnancy, along with the additional possibility that the pain may be directly attributable to the pregnancy. Labor is rarely described as abrupt in onset, is usually colicky in nature, and is often associated with other symptoms or signs, such as blood-tinged vaginal discharge. When there is doubt regarding the cause of the pain, observation and serial examinations of the cervix for evidence of change are helpful. Musculoskeletal pain is common, and its likelihood increases as pregnancy advances, owing to weight gain, the loosening of connective tissues with the hormonal changes of pregnancy, and the shift forward in the woman’s center of gravity. Pyelonephritis is a concern with an abrupt onset of back pain, but it is unlikely in this patient, given the reported location of the pain and the absence of fever, chills, urinary frequency, and dysuria.

The patient returned to her obstetrician the next day with worsening pain located in the middle-to-lower back, now with radiation to the upper abdomen. She reported an episode of vomiting that morning. She still had no fever, chills, sweats, or urinary symptoms and reported no changes in bowel function and no vaginal bleeding or headache. She was referred to the emergency room for further evaluation.
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On physical examination, the blood pressure was 159/91 mm Hg, pulse 95 beats per minute, and temperature 36.6°C (97.9°F). The abdominal examination showed a gravid uterus and epigastric tenderness without rebound or guarding. There was no palpable mass or hepatosplenomegaly and no tenderness at the costovertebral angle.

More information is needed regarding the nature of the pain. Colicky pain could be intestinal, renal, biliary, or uterine in origin. For the first three of these sources, the presentation in a pregnant woman would not be expected to differ from that in a nonpregnant woman. Although uterine contractions are relatively easy to detect, are very common, and occur with increasing frequency as pregnancy progresses, premature labor is notoriously difficult to distinguish from contractions unrelated to labor. Noncolicky pain could reflect an adnexal condition, such as ovarian torsion.

Design
Fifty-three patients (45 women and 8 men) with diabetes were studied in a randomized controlled clinical trial and assigned into two groups: sour tea (Hibiscus sabdariffa) and black tea. Patients were instructed to consume one glass of the decoction (two spoonfuls of blended tea in a sachet weighing 2 g, placed in one glass of boiled water and boiled for 20–30 minutes) with 5 g sugar, twice daily; they also were instructed not to drink any other types of tea during the study. Tea consumption was continued for 1 month. Fasting blood samples were obtained before and after intervention, testing HDL, LDL, triglycerides, Apo-A1, Apo-B1-00, and Lp(a). 20.8% of the patients were only on diet therapy for their diabetes and the rest were either on insulin or oral hypoglycemic agents.

Key Findings
Hibiscus consumption reduced most of the lipids and lipoproteins and increased HDL, but black tea consumption only increased HDL.

Hibiscus consumption reduced most of the lipids and lipoproteins and increased HDL, but black tea consumption only increased HDL. The sour tea group averaged a 7.6% decrease in total cholesterol, 8.0% decrease in LDL, 14.9% decrease in triglycerides, 3.4% decrease in Apo-B100, 16.7% increase in HDL, 4.2% increase in Apo-A1, and no change in Lp(a). The black tea group averaged a 13% increase in HDL but did not see a significant change in other measures.

Commentary
Previous studies have been done on Hibiscus and dyslipidemia. Lin, et al, showed that drinking Hibiscus tea for 4 weeks reduced total cholesterol by 8.3%–14.4%.1 Animal studies have shown that rats with diabetes had a positive effect with Hibiscus on glucose and lipids.2 I reported on the effectiveness of Hibiscus tea in reducing systolic blood pressure in a recent column.

Hibiscus sabdariffa, or sour tea, is used in many parts of the world to make both cold and hot drinks. We might know it in English by the name Hibiscus or red sorrel. In Arabic it is called karkade, and in Iran it is mainly known as sour tea. It contains many constituents including alkaloids, L-ascorbic acid, anisaldehyde, anthocyanin, beta carotene, beta sitosterol, citric acid, cyaniding-3, rutinoside, delphinidin, galactose, gossypetin, hibiscetin, mucopolysaccharide, pectin, protocatechuic acid, polysaccharide, quercetin, stearic acid, and wax. The medicinal parts of the plant include the flower, calyx or sepal, leaves, stems, and seeds. The extracts of the sepal contain significant amounts of vitamin C, anthocyanins, and polyphenols, as well as the highest concentration of water-soluble antioxidants. Traditionally, it has been used in folk medicine for several health issues including high blood pressure, liver diseases, loss of appetite, circulation, and as a gentle laxative and diuretic.

In addition to its appealing flavor, smell and brightly colored appearance, we can add this simple tea to our medicinal options for hyperlipidemia and hypertension.

Thorough review of the recent literature yields very little in terms of in vivo validation of mechanisms of CD44 signaling in HNSCC. This represents a deficiency in the current state of scientific knowledge. Most work to date related to CD44 signaling in HNSCC has involved analysis of in vitro data from HNSCC cell lines or relies on inferences from immunohistochemical analysis of patient tissue specimens. For breast cancer, there are in vivo data on CD44 signaling. Co-expression of CD44 v10 and CD44s through transfection of nonmalignant human breast epithelial cells was shown to promote tumorigenesis in athymic nude mice, but not for nontransfected or vector-only transfected parental cells.50 Furthermore, we can infer the importance of CD44 on HNSCC progression in vivo from studies of CD44 as a cancer stem cell marker in HNSCC, in which CD44-positive enriched HNSCC cells had greater tumorigenicity in nude mice, compared with CD44-negative HNSCC cells. Nonetheless, studies of CD44-mediated migration, metastasis, and chemoresistance or radiation resistance in an in vivo model of HNSCC are currently lacking in the literature.

Conclusion

In summary, an accumulating body of evidence highlights the important role of HA and CD44 signaling in HNSCC progression. In Figure 2 we present a model summarizing our current understanding of the role of HA and CD44 interaction with oncogenic signaling pathways to promote tumor progression and chemoresistance in HNSCC. Subsequent to the HA/CD44 interaction that recruits and forms a CD44-EGFR-LARG multimolecular complex, multiple downstream signaling pathways are activated, and cross-talk among Ras, RhoA, ROK, and PI-3 kinase can occur, further promoting diverse tumor progression behaviors. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment, early detection, and prevention for this deadly disease. Research to date suggests that targeted inhibition of HA/CD44-mediated signaling combined with conventional chemotherapy agents may be an efficacious strategy, one that should be pursued to improve the future treatment of advanced HNSCC.

Hyaluronan-stimulated intracellular Ca2+ mobilization mediates important components of the CD44 signaling pathways. Some of these Ca2+-mediated pathways may be mediated by the Ca2+ binding protein calmodulin. It is known that calmodulin is involved in the activation of several important enzymes, including calcium/calmodulin-dependent protein kinase type II (CaMKII), a ubiquitous serine/threonine protein kinase. In HNSCC cells, CaMKII activation by HA/CD44-mediated Ca2+ mobilization results in the phosphorylation of diverse substrates that promote various cell functions, including motility, cell cycle progression, and proliferation. CaMKII phosphorylates the cytoskeletal protein, filamin. These HA/CD44-mediated effects on CaMKII and filamin lead to cytoskeleton reorganization and promote tumor cell migration. Wang et al49 linked HA/CD44-dependent CaMKII activity to topoisomerase II regulation in HNSCC cells. Topoisomerase II is a critical regulator of DNA topology and function. Hyaluronan treatment promoted CaMKII-dependent topoisomerase II phosphorylation, resulting in enhancement of topoisomerase II activity and decreased cytotoxicity of etoposide (a topoisomerase II poison).These HA/CD44-mediated effects on CaMKII and topoisomerase II activity enhanced tumor cell survival.

Another important effector of RhoA pathway signaling is Rho kinase (ROK). Activated ROK is known to phosphorylate a number of cytoskeletal proteins, such as myosin phosphatase and adducin, that are highly involved in tumor migration and to promote the secretion of MMPs involved in tumor invasion. Torre et al recently showed that HA/CD44 interaction increased ROK activity in HNSCC cells. Hyaluronan also promoted Rho kinase-mediated myosin phosphatase phosphorylation, resulting in enhanced tumor cell migration, and it increased activated MMP-2 and MMP-9 secretion.

RhoA/Ca2+ Signaling-Regulated Chemoresistance

HA/CD44 interaction has been shown to promote resistance to multiple chemotherapeutic agents in HNSCC, including cisplatin, methotrexate, doxorubicin (Adriamycin), and etoposide.38, 40, 49 Several cell signaling mechanisms appear to promote CD44-mediated chemoresistance in HNSCC, including EGFR-related signaling pathways (as already described here).38 Recent work by our groupsuggests that regulation of Ca2+ may also affect chemoresistance (unpublished data). The median inhibitory concentration IC50 for the chemotherapy agent methotrexate in the HNSCC cell line SCC4 is dependent on both the Ca2+ level and the presence of HA. SCC4 cells grown in 1.2 mmol/L Ca2+ medium had greater resistance to methotrexate than cells grown in low-Ca2+ medium, and the IC50 was increased in the presence of HA at both Ca2+ concentrations.

Phospholipase C and RhoA signaling, which mediate intracellular Ca2+ levels, has been shown to play roles in mediating chemoresistance in HNSCC. Wang et al demonstrated that HA-mediated cisplatin, methotrexate, and doxorubicin resistance could be eliminated with inhibition of PLC. Torre et al found that combined ROK and PI-3 kinase inhibition resulted in a synergistic prosurvival effect in the presence of cisplatin. Thus, HA-mediated chemoresistance in HNSCC may involve multiple pathways, including RhoA-mediated Ca2+ signaling.

Ankyrin is a membrane-associated cytoskeletal protein that directly binds CD44.10 This CD44-ankyrin interaction causes cytoskeleton activation. Hyaluronan binding to CD44 promotes colocalization of CD44 and ankyrin in cholesterol-containing lipid rafts, and this colocalization appears to be a key mechanism in regulating HA-mediated cytoskeleton function and tumor cell-specific behaviors (eg, cell survival, growth, and migration). Ankyrin- and CD44-containing lipid rafts have been documented in three different tumor cell lines (breast cancer, ovarian cancer, and HNSCC).10

Up-regulated expression of the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins is seen in HNSCC and is associated with poor prognosis.45 ERM proteins are linkers between membrane molecules such as CD44 and the cytoskeleton. Ezrin is a key regulator of tumor metastasis. CD44 interacts with ERM proteins and with merlin, a related protein. Loss of merlin results in increased HA/CD44-mediated tumorigenesis, and overexpression of merlin diminishes tumor cell growth. In HNSCC, moesin appears to interact with CD44 to degrade the ECM at the invasive front of oral cancers. Both the level of expression and the subcellular localization (cytoplasmic) of ERM proteins are indicators of clinical outcome in HNSCC. Higher expression and cytoplasmic localization of ERM proteins are indicators of poorer survival in HNSCC. Osteopontin, a ligand for CD44, colocalizes with CD44 and ezrin in fibroblasts, metastatic breast cancer cells, and HNSCC.
RhoA-Regulated PLC, ROK, and Ca2+ Signaling and Cytoskeleton Activation

Similar mechanisms for CD44-mediated Ca2+ mobilization pathways to promote tumor migration appear to exist in HNSCC. In HNSCC, Bourguignon et al37 reported that CD44 physically associates in a multimolecular complex with LARG and EGFR. HA/CD44 interaction induces LARG-specific RhoA signaling. LARG molecules isolated from HNSCC cells were found to function as a GDP/GTP exchange factor for RhoGTPases, and the basal rate of bound GTP increased at least 2.4-fold with the addition of HA.

To establish a linkage between HA/CD44-mediated LARG-RhoA signaling and intracellular Ca2+ regulation, one member of the PLC family, PLCε, was isolated from HNSCC cells with GDP- or GTP-loaded forms of RhoA-GST-conjugated beads. PLCε-RhoA interaction was found to be GTP-dependent. LARG-activated RhoA was shown to stimulate both the PLCε-mediated IP3 production and the IP3 receptor-triggered intracellular Ca2+ mobilization in HNSCC cells. HNSCC cells incubated with Fura-2/AM were treated with HA with or without pretreatment with inhibitors of PLC and IP3 receptor; fluorescence spectrophotometry demonstrated a rise in intracellular Ca2+ with HA treatment, but not in the presence of pretreatment with various inhibitors.40 These findings suggest that Ca2+ signaling in HNSCC cells involves both HA/CD44-dependent and RhoA/PLC/IP3 receptor-regulated processes.

One of the key downstream molecular effectors in EGFR-mediated cell survival is PI-3 kinase. PI-3 kinase is capable of catalyzing the conversion of phosphatidylinositol-3,4-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which subsequently results in the activation of AKT. AKT has 3 subtypes: AKT-1, AKT-2, and AKT-3. HA/CD44 signaling in HNSCC activates all three AKT subtypes. Activated AKT down-regulates the cell’s apoptotic potential, thus promoting increased proliferation and tumor cell survival. Torre et al reported that HA/CD44 interaction increased PI-3 kinase activity in HNSCC cells; HA also promoted PI-3 kinase-mediated AKT phosphorylation, resulting in increased tumor cell survival.
CD44-EGFR Signaling and Chemoresistance

HA/CD44 signaling not only appears to promote EGFR-mediated prosurvival effects, but also leads to increased chemoresistance in HNSCC through EGFR signaling pathways. Cisplatin is the most common anticancer drug used today for the treatment of HNSCC. Wang et al reported that HA can promote cisplatin resistance in several HNSCC cell lines; however, the HA-mediated cisplatin resistance could be abolished with inhibitors of EGFR and MAPK.38 The mechanism of action of cisplatin is thought to involve apoptosis-induced cell death. Because AKT suppresses apoptosis, its activation by PI-3 kinase has been suggested to play a key role in cisplatin resistance. Torre et al39 demonstrated that LY-294002, an inhibitor of PI-3 kinase, was capable of blocking HA-mediated cisplatin resistance. Taken together, these reports suggest that HA/CD44 can promote cisplatin resistance in HNSCC through EGFR signaling pathways.

Interaction of HA and CD44 Promotes Cytoskeleton Activation, Migration, Invasion, and Chemoresistance Pathways in HNSCC

A hallmark of all solid malignancies is the ability to invade and/or metastasize to distant sites. Tumor cells possess altered signaling pathwaysthat lead to cytoskeleton activation and migration. Additionally, tumor cells secrete extracellular factors, such as matrix metalloproteinases, to allow breakdown and invasion of the surrounding ECM. Both HA, which is a major component of the ECM, and its major ligand receptor, CD44, have been studied for their role in promoting tumor progression properties, including migration and invasion. The mechanisms of how HA and CD44 interact to promote cytoskeleton activation, migration, and invasion were initially elucidated in several cancer and noncancer models.

RhoA signaling appears to be a critical pathway through which HA/CD44 interaction mediates cytoskeleton activation. One of several important mechanisms used by RhoA in the regulation of cellular functions is through alteration of intracellular Ca2+ levels. Phospholipase C (PLC) is a key mediator in intracellular Ca2+ mobilization. When activated by RhoA, PLCs first hydrolyze PIP2 into inositol trisphosphate (IP3), resulting in Ca2+ release from intracellular stores. This Ca2+ release promotes various cell functions, including cytoskeleton activation, cell cycle progression, and proliferation. Another key RhoA pathway effector is Rho kinase (ROK), which has been shown to regulate several cytoskeletal proteins (such as myosin light chain phosphatase) that are highly involved in tumor migration and to promote the secretion of MMPs, which degrade the ECM during tumor invasion.

HA/CD44 interaction has been shown to be tightly coupled with intracellular Ca2+ mobilization pathways and with RhoA pathways in many different cells. CD44 and RhoA are physically associated in metastatic breast cancer cells, and ROK was found to play a key role in CD44-ankyrin interaction and in RhoA-mediated breast cancer oncogenic signaling. In endothelial cells, HA treatment promoted CD44 interaction with ROK, leading to IP3 receptor-mediated Ca2+ mobilization and migration.9 In keratinocytes, HA-mediated Ca2+ mobilization promoted cortactin-cytoskeleton function, leading to adhesion and differentiation.44 CD44 is linked to ankyrin and has been shown to activate MMP-9 during active migration processes in metastatic breast cancer cells.